No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Isolation and characterization of a human cDNA encoding uracil-DNA glycosylase
Abstract
DNA repair of genetic information is an essential defense mechanism, which protects cells against mutation and transformation. The biochemistry of human DNA repair is in its beginning stages. Our research has concentrated on the enzymes involved in the removal of atypical bases from DNA. We present information on the identification and characterization of a cDNA isolate encoding uracil-DNA glycosylase. Uracil-DNA glycosylase was purified to homogeneity from HeLa S3 cells and used to generate polyclonal antibodies. These antibodies were in turn used to isolate a uracil-DNA glycosylase specific cDNA from a human T cell (Jurkat) lambda-gt11 library. The identity of this 1.25 kb cDNA was verified using in vitro transcription and translation systems to generate specific uracil-DNA glycosylase activity. Sequence data revealed a 327 amino acid open reading frame, which encodes a protein with a predicted molecular weight of 35351. No significant amino acid homology was found between this human uracil-DNA glycosylase and the glycosylases of yeast, Escherichia coli, herpes simplex virus, or a recently identified 26,000 Da species of human uracil-DNA glycosylase. This apparent lack of homology prompted an investigation of uracil-DNA glycosylase in a variety of eukaryotic species. Western analysis demonstrated the presence of a 36 kDa uracil-DNA glycosylase protein in human fibroblast, human placental and Vero cell extracts. Interestingly, these antibodies did not detect glycosylase protein in Chinese hamster ovary (CHO) or mouse NIH3T3 fibroblast cells. Under conditions of reduced stringency, Southern blot analysis of BamHI digested DNA from human fibroblasts, human placental cells and Vero cells revealed common 12 kb and 3 kb fragments. In contrast, using the same reduced stringency protocol, 6 and 8 kb fragments for CHO and NIH3T3 DNA were seen, respectively, as well as a common 3 kb fragment. Under more stringent wash conditions, the common 3 kb band was absent in all samples analyzed, and no hybridization signal was detected from DNA of hamster or mouse origin. The lack of immunological reactivity between the human uracil-DNA glycosylase and the rodent forms is therefore reflected at the genetic level as well. This distinction in human and CHO hybridization patterns enabled us to localize this human uracil-DNA glycosylase cDNA to chromosome 5 by somatic cell hybridization.
Supplementary resource (1)
... The restriction map of the cDNA is in accord with our Southern blot analysis of human genomic DNA digested with either EcoRI or Pst I (5). In contrast, a recently described putative mitochondrial human UDG cDNA and a second human nuclear UDG cDNA did not contain Xba I sites and did contain multiple Pst I sites (11,12). The complete nucleotide sequence of the cDNA was then determined (Fig. 4). ...
... USA 88 (1991) calculated pI of 8.5 agrees with the pI of the purified human placental UDG (G.S. & M.A.S., unpublished work). Comparison of this sequence with all other known UDG sequences demonstrated no significant homology (9,11,12,15,16). However, a search of the GenBank data base revealed complete (>99%) homology with nucleic acid and amino acid sequences for the 37-kDa subunit of the glycolytic enzyme G3PD (17). ...
We have isolated and characterized a plasmid (pChug 20.1) that contains the cDNA of a nuclear uracil DNA glycosylase (UDG) gene isolated from normal human placenta. This cDNA directed the synthesis of a fusion protein (Mr 66,000) that exhibited UDG activity. The enzymatic activity was specific for a uracil-containing polynucleotide substrate and was inhibited by a glycosylase antibody or a beta-galactosidase antibody. Sequence analysis demonstrated an open reading frame that encoded a protein of 335 amino acids of calculated Mr 36,050 and pI 8.7, corresponding to the Mr 37,000 and pI 8.1 of purified human placental UDG. No homology was seen between this cDNA and the UDG of herpes simplex virus, Escherichia coli, and yeast; nor was there homology with the putative human mitochondrial UDG cDNA or with a second human nuclear UDG cDNA. Surprisingly, a search of the GenBank data base revealed that the cDNA of UDG was completely homologous with the 37-kDa subunit of human glyceraldehyde-3-phosphate dehydrogenase. Human erythrocyte glyceraldehyde-3-phosphate dehydrogenase was obtained commercially in its tetrameric form. A 37-kDa subunit was isolated from it and shown to possess UDG activity equivalent to that seen for the purified human placental UDG. The multiple functions of this 37-kDa protein as here and previously reported indicate that it possesses a series of activities, depending on its oligomeric state. Accordingly, mutation(s) in the gene of this multifunctional protein may conceivably result in the diverse cellular phenotypes of Bloom syndrome.
... This might imply an increase in accessibility due to "wobble" formation when uracil forms a base pair with guanine as indeed is the case in TG base pairs (Hunter, 1992). Two different cDNAs claimed to encode human uracil-DNA glycosylase have been isolated by other groups (Vollberg et al., 1989;Muller & Caradonna, 1991), and this has resulted in considerable confusion as to which of the cDNAs encodes the major (nuclear) form of UDG in human cells. We have recently demonstrated that UNG15 encodes UDG which is mainly located in nuclei, but a minor fraction is transported to mitochondria . ...
... The specific activity of this gene product is several orders of magnitude lower than that of the UNG protein and may represent a cryptic activity. The functional significance of the cDNA isolated by Muller and Caradonna (1991) is also still not clear. This gene encodes a protein with a significant similarity to the conserved region of the cyclin protein family, and thus bears no obvious resemblance to the structurally conserved UDG family. ...
We have expressed a human recombinant uracil-DNA glycosylase (UNG delta 84) closely resembling the mature form of the human enzyme (UNG, from the UNG gene) in Escherichia coli and purified the protein to apparent homogeneity. This form, which lacks the first seven nonconserved amino acids at the amino terminus, has properties similar to a 50% homogeneous UDG purified from human placenta except for a lower salt optimum and a slightly lower specific activity. The recombinant enzyme removed U from ssDNA approximately 3-fold more rapidly than from dsDNA. In the presence of 10 mM NaCl, Km values were 0.45 and 1.6 microM with ssDNA and dsDNA, respectively, but Km values increased significantly with higher NaCl concentrations. The pH optimum for UNG delta 84 was 7.7-8.0; the activation energy, 50.6 kJ/mol; and the pI between 10.4 and 10.8. The enzyme displays a striking sequence specificity in removal of U from UA base pairs in M13 dsDNA. The sequence specificity for removal of U from UG mismatches (simulating the situation after deamination of C) was essentially similar to removal from UA matches when examined in oligonucleotides. However, removal of U from UG mismatches was in general slightly faster, and in some cases significantly faster, than removal from UA base pairs. Immunofluorescence studies using polyclonal antibodies against UNG delta 84 demonstrated that the major fraction of UNG was located in the nucleus. Furthermore, > 98% of the total uracil-DNA glycosylase activity from HeLa cell extracts was inhibited by the antibodies, indicating that the UNG protein represents the major uracil-DNA glycosylase in the cells.
... In humans, four putative uracil-DNA glycosylases have been identified. These include the following: 1) a 29,000 molecular weight protein that is highly conserved in nature (10); 2) a cyclin-like protein (UDG2) (11); 3) thymine-DNA glycosylase (12); and 4) a recently identified isoform of the highly conserved uracil-DNA glycosylase (UDG1A) (13). At present an in vivo role for many of these proteins is unclear. ...
... At present an in vivo role for many of these proteins is unclear. For example, the cyclin-like UDG2 generates uracil-DNA glycosylase activity in vitro and specifically interacts with the uracil-DNA glycosylase inhibitor protein (11,14). However, we do not know how or when this (uracil-removing) activity is required by the cell. ...
We have previously demonstrated that human cells contain multiple forms of uracil-DNA glycosylase (Caradonna, S. J., Ladner, R., Hansbury, M., Kosciuk, M., Lynch, F., and Muller, S. J. (1996) Exp. Cell Res. 222, 345-359). One of these is an Mr 29,000 processed form of the highly conserved uracil-DNA glycosylase (UDG1) located in the mitochondria. The others are located in the nucleus and migrate as a group of at least three distinct bands within the 35,000-37,000 molecular weight range. In this report, we perform a detailed characterization of the Mr 35,000-37,000 purified proteins. To accomplish this, uracil-DNA glycosylases were affinity purified from HeLa cell nuclear extracts. The proteins were separated by SDS-PAGE, and their identities were verified by renaturation and activity assays. The three protein bands were individually digested with cyanogen bromide, and the resulting peptide fragments were analyzed by direct amino acid sequencing. Peptide sequence, derived from each band, was identical and corresponded to a recently identified isoform of UDG1. This isoform (UDG1A) has a unique 44-amino acid N-terminal region and a C-terminal region that is identical to UDG1. To begin to study the signals required for nuclear targeting, the N-terminal regions of UDG1 and UDG1A were isolated and cloned into pEGFP-N2 to generate fusions with a red-shifted variant of green fluorescent protein (GFP). When these constructs were transfected into NIH3T3 cells, UDG1/pEGFP was targeted to the mitochondria, and UDG1A/pEGFP was targeted to the nucleus. Further studies, using deletion mutants, demonstrate that the nuclear localization signal resides within the first 20 amino acids of UDG1A. To investigate the possibility that the heterogeneity observed on SDS-PAGE results from post-translational modification(s), the UDG/pEGFP fusion constructs were transfected into NIH3T3 cells, and the cells were metabolically labeled with [32P]orthophosphate. Results from these experiments show that UDG1A is a phosphoprotein. Subsequent phosphoamino acid analysis revealed that UDG1A is phosphorylated on both serine and threonine residues. As a final characterization, RNase protection assays were performed to examine expression of each of these isoforms. These studies demonstrate that UDG1A is expressed in a wide variety of cell types and that message levels are elevated in transformed cells.
... UNG1 and UNG2 have unique N-terminal regions required for subcellular sorting (Nilsen et al., 1997; Otterlei et al., 1998), while the 3834 © European Molecular Biology Organization structurally and biochemically well-characterized catalytic domain (Mol et al., 1995; Slupphaug et al., 1996; Parikh et al., 1998) is common for the two forms. Other uracil-DNA glycosylases have also been reported, including a thymine(uracil)-DNA glycosylase (TDG) with a strong preference for T or U mispaired with G (Nedderman and Jiricny, 1994), a cyclin-like uracil-DNA glycosylase (Muller and Caradonna, 1991) and a very recently reported uracil-DNA glycosylase (SMUG1) that like UNG proteins prefers uracil in single-stranded DNA as substrate (Haushalter et al., 1999). It has not yet been determined whether the different uracil-DNA glycosylases have distinct or overlapping physiological functions. ...
... We also demonstrate that removal of incorporated dUMP is carried out mainly by UNG2, since neutralizing antibodies to UNG2 efficiently protected uracil from excision. This would seem to rule out a major function for TDG, which actually removes uracil more efficiently than thymine (Neddermann and Jiricny, 1994), the reported cyclin-related uracil-DNA glycosylase (Muller and Caradonna, 1991) and SMUG1 (Haushalter et al., 1999) in the removal of incorporated uracil residues. The rapid removal of incorporated uracil is consistent with the previous observation that incorporation of dUMP into growing DNA strands in isolated nuclei from S phase cells results in fragmentation of DNA to sizes smaller than normal Okazaki fragments within 1 min after the incorporation of dUMP (Wist et al., 1978). ...
Base excision repair (BER) is initiated by a DNA glycosylase and is completed by alternative routes, one of which requires proliferating cell nuclear antigen (PCNA) and other proteins also involved in DNA replication. We report that the major nuclear uracil-DNA glycosylase (UNG2) increases in S phase, during which it co-localizes with incorporated BrdUrd in replication foci. Uracil is rapidly removed from replicatively incorporated dUMP residues in isolated nuclei. Neutralizing antibodies to UNG2 inhibit this removal, indicating that UNG2 is the major uracil-DNA glycosylase responsible. PCNA and replication protein A (RPA) co-localize with UNG2 in replication foci, and a direct molecular interaction of UNG2 with PCNA (one binding site) and RPA (two binding sites) was demonstrated using two-hybrid assays, a peptide SPOT assay and enzyme-linked immunosorbent assays. These results demonstrate rapid post-replicative removal of incorporated uracil by UNG2 and indicate the formation of a BER complex that contains UNG2, RPA and PCNA close to the replication fork.
... Meanwhile, the cyclin activation and the repression of CKIs proteins can be regulated by EGFR signal pathway (4). Cyclin O (CCNO) is a novel cyclin family protein containing a cyclin-like domain, consisting of three exons located on chromosome 5q11 encoding a 1,053 nt mRNA and a 350-aminoacid protein (5). According to reports, CCNO is produced during the S phase and colocalizes with the replication foci of proliferating cell nuclear antigen protein before being destroyed during the S/G2 transition (6). ...
Background:
Cyclin O (CCNO) is a novel cyclin family protein containing a cyclin-like domain, which plays a role in cell cycle regulation. Recent research suggests that inhibition of CCNO leads to cell apoptosis in gastric cancer, cervical squamous cell carcinoma, and post-operative lung cancer.
Methods:
The protein expression and signal transduction were detected by Western blot (WB) and immunohistochemistry (IHC). Overexpression or lacking CCNO stable cell lines were transfected with lentiviruses and selected with puromycin. The tumor behaviors of lung adenocarcinoma (LUAD) cells were assessed: cell proliferation by 5-Ethynyl-2'-deoxyuridine (EdU) staining and Cell Counting Kit-8 (CCK8) assay, cell cycle and by flow cytometry analysis, and migration and invasion using wound healing and Transwell system. Co-immunoprecipitation was used to detect protein-protein interactions. Xenograft models for evaluating tumor growth and anti-tumor drug efficacy.
Results:
A higher expression of CCNO was observed in LUAD cancer tissues and predicted the overall survival of LUAD patients. Moreover, CCNO expression was negatively correlated with cancer cell proliferation, migration, and invasion. Co-immunoprecipitation and western blot indicated that CCNO interacted with CDK13 to promote cancer cell proliferation signaling activation. Furthermore, CCNO promoted tumor cell growth and cetuximab resistance in vivo, and a CDK13 inhibitor effectively inhibited the oncological effect of CCNO.
Conclusions:
The current study suggests that CCNO may be a driver in the development of LUAD and that its function is related to CDK13 interaction that promotes proliferation signaling activation.
... While S. cerevisiae only has one (Ung1), mammals have four (UNG, SMUG1, TDG, MBD4). Mammalian glyceraldehyde-3phosphate dehydrogenase (GAPDH) and cyclin O (CCNO) have also been reported to have uracil excision activity (203)(204)(205). These activities have not been characterized beyond these initial reports, though a 2015 study reported that while GAPDH binds strongly to abasic sites, no uracil excision activity was observed (206). ...
Deoxyribonucleic acid (DNA) encodes the information necessary for all functions of life. Despite the critical importance of DNA, its chemical structure is susceptible to chemical and physical alterations including base damage and single‐ and double‐strand break (DSB) induction. Cellular DNA damage results from both environmental exposures and endogenous sources from normal cellular metabolism. DNA damage in both the nuclear and mitochondrial genomes is associated with several human diseases including the development of cancer. The biological consequences of DNA damage are largely determined by a cell's DNA repair capabilities and the interaction of damaged DNA with the replication and transcription machinery. Technologies exist for the detection and measurement of cellular DNA damage in varying contexts.
Key Concepts
• Estimated rates of induction of various deoxyribonucleic acid (DNA) lesions, including spontaneous hydrolysis and oxidative DNA damage, have been determined.
• Potential biological consequences of DNA damage include physiological conditions such as cancer, neurodegenerative diseases, heart disease and ageing.
• Various exogenous and endogenous sources induce DNA base lesions and strand breaks that can be detected by molecular and biochemical methods.
• Incorporation of damaged deoxyribonucleotides during DNA synthesis can occur due to errors committed by DNA polymerases and/or disruptions of nucleotide pool balance.
• Many of the features resulting from DNA packing into chromatin, including DNA–protein interactions and sequence context, greatly impact how damage is distributed within the genome.
• Mutational signatures of cancers arise from context‐dependent DNA damage and can be attributed to specific types of DNA damage.
• DNA is often the target of chemotherapeutic drugs used to treat cancer, resulting in tumour cell death.
• Mitochondrial DNA damage can increase reactive oxygen species, leading to cell death and in certain instances give rise to mutations that contribute to cancer phenotypes.
... However, an antibody that is specific only to this recently identified type of UDGase also cross reacts with ceU extracts from simian species, but not rodents, indicating that it is a recently acquired activity in evolutionary terms. This particular type of new UDGase is also found to be a ceU cycle dependent activity (MuUer and Caradonna, 1991;MuUer and Caradonna, 1993). This proliferative dependency of expression has also been reported for other UDGases (Yamamoto and Fujiwara, 1986;Hernandez and Gutierrez, 1987;Cool and Sirover, 1989;Slupphaug et al., 1991;Dudley et a/., 1992;Meyer-Siegler et a/., 1992;Bones, 1993;Weng and Sirover, 1993 Upton et a/., 1993) has further increased interest in the apparently indispensable role of this enzyme in any organism that carries a DNA genome. ...
The work presented in this thesis describes experiments carried out in order to determine the three-dimensional structure of a DNA repair enzyme, uracil-DNA glycosylase. An open reading frame, UL2, in the herpes simplex virus type 1 genome, is known to encode a uracil-DNA glycosylase. By sequence homology, there are three candidate start codons which might express a functional uracil-DNA glycosylase. Expression from two of these was attempted in Escherichia coli, using plasmids designed for high level production of recombinant proteins. The second candidate start codon produces high levels of a soluble, functional uracil-DNA glycosylase in Escherichia coli both in a native form, and as part of a fusion protein. Both the fusion and the native form of the enzyme have been purified to apparent homogeneity, as has a recombinantly expressed insoluble Escherichia coli uracil-DNA glycosylase. Preliminary attempts were made at deriving structural and functional information from the soluble, native recombinant herpes simplex enzyme with the use of circular dichroism. This form of uracil-DNA glycosylase has subsequently been crystallised in two ways, firstly as the free enzyme, and secondly in a complex with a single stranded DNA oligonucleotide. Extensive optimisation of the crystallisation parameters have been carried out in conjunction with modifications to the original purification protocol, and large, single crystals of both free, and DNA bound forms, suitable for X-ray diffraction studies are now readily reproduced. A systematic search for isomorphous heavy atom derivatives has been carried out for both types of crystal, and preliminary phases have been obtained for the DNA-bound form of the enzyme. This has enabled the calculation of an electron density map in which protein secondary structure features can be located. Improvement of this map will reveal the molecular structure of the enzyme/ DNA complex.
... In humans; the UNG gene is what encodes for UNG, the major cellular form of UDG in humans (13.). Alternative promoter usage and splicing of this gene leads to two different isoforms: the mitochondrial UNG1 and the nuclear UNG2 cyclin-like human Uracil DNA glycosylase (12). In further depth of UDG's excision mechanism, it starts its base ex-traction repair that ca uses the expulsion of Uracil from the DNA. ...
DNA replication, repair and recombination processes are important for genome stability. DNA Flap endonucleases are a class of nucleolytic enzymes that are important in maintaining these processes. They act as 5'- 3' exonucleases and structure-particular endonu-cleases on specific DNA structures (1)(5). Uracil-DNA Glycosylase (UDG) and its superfamily are enzymes involved DNA repair, in particular base excision repair. This review focuses on DNA flap endonucleases; we also associated this with our practical investigation in purification, expression and quantification of His-tagged Uracil-DNA Glycosylase. To purify UDG, we utilized different chromatography methods and for purity, comparisons were made through SDS-PAGE analysis. In a pull down assay, we tested for expression of UDG in a western blot and tested UDG’s activity on Uracil DNA when inhibited via Uracil Glycosylase Inhibitor (Ugi).Purification of UDG via all three chromatography methods appeared successful and similar levels of purity were achieved, yet Size Exclusion Chromatography purified best. UDG ex-pression was positive. UGI inhibited UDG activity in cleaving out Uracil from DNA, in which resulted in a shorter DNA fragment (kb).
... Subsequent cloning and sequencing of UDG genes from Saccharomyces cerevisiae (5), human (6) and other sources established that UDG is a highly conserved protein. Interestingly, the organisms also contain a second group of UDGs consisting of a diverse group of proteins such as the cyclin-like UDG (7,8), dsUDG (9) and glyceraldehyde 3 phosphate dehydrogenase (10). None of the UDGs studied so far require metal ions or other cofactors for its activity (11)(12)(13)(14). ...
... Subsequent cloning and sequencing of UDG genes from Saccharomyces cerevisiae (5), human (6) and other sources established that UDG is a highly conserved protein. Interestingly, the organisms also contain a second group of UDGs consisting of a diverse group of proteins such as the cyclin-like UDG (7,8), dsUDG (9) and glyceraldehyde 3 phosphate dehydrogenase (10). None of the UDGs studied so far require metal ions or other cofactors for its activity (11)(12)(13)(14). ...
Uracil-DNA glycosylase (UDG), a key highly conserved DNA repair enzyme involved in uracil excision repair, was discovered
in Escherichia coli. The Bacillus subtilis bacteriophage, PBS-1 and PBS-2, which contain dUMP residues in their DNA, express a UDG inhibitor protein, Ugi which binds
to UDG very tightly to form a physiologically irreversible complex. The X-ray analysis of the E.coli UDG (EcUDG)-Ugi complex at 3.2 Å resolution, leads to the first structure elucidation of a bacterial UDG molecule. This structure
is similar to the enzymes from human and viral sources. A comparison of the available structures involving UDG permits the
delineation of the constant and the variable regions of the molecule. Structural comparison and mutational analysis also indicate
that the mode of action of the enzyme from these sources are the same. The crystal structure shows a remarkable spatial conservation
of the active site residues involved in DNA binding in spite of significant differences in the structure of the enzyme-inhibitor
complex, in comparison with those from the mammalian and viral sources. EcUDG could serve as a prototype for UDGs from pathogenic prokaryotes, and provide a framework for possible drug development
against such pathogens with emphasis on features of the molecule that differ from those in the human enzyme.
... Cyclin O (CCNO), a cyclin-like DNA glycosylase that removes the cytosine deamination or misincorporated uracil on DNA, consists of three exons located on chromosome 5q11 encoding a1, 053-bpcDNA, and a 350-amino-acid protein. 8,9 Nowadays, CCNO has been referred to as a cyclin-like protein containing two cyclin box regulatory elements predicted to function as protein-binding domains. 9,10 It has been reported that CCNO is expressed in the S phase, colocalizing with proliferating cell nuclear antigen protein at the replication foci, while degraded at the S/G2 transition stage. ...
Purpose:
Recently, Cyclin O (CCNO) has been reported to be a novel protein of the cyclin family. However, the clinical significance and functional roles of CCNO in human cancer, including gastric cancer (GC), remain largely unexplored. In this study, we investigated the clinical and functional roles of CCNO in GC.
Methods:
We analyzed CCNO expression patterns in GC patients. To investigate the role of CCNO in malignancy of GC, we used lentivirus-delivered short hairpin RNA to knockdown CCNO expression in GC cell lines. Then multiparametric high-content screening and MTT incorporation assay were used to assess the cell proliferation capability. Cell apoptosis was detected by flow cytometry and Caspase 3/7 assays. Furthermore, the effect of CCNO on tumorigenicity of GC was also determined in vivo. Finally, microarray analysis was performed to elucidate the molecular mechanisms by which shCCNO inhibited the malignancy of GC cells.
Results:
The analysis from The Cancer Genome Atlas database revealed elevated CCNO mRNA expression in GC tissue than in the adjacent normal tissue. Immunohistochemical studies also showed that stronger cytoplasmic staining of CCNO was detected in GC tissues. Downregulation of CCNO in GC cells efficiently, through infection with the lentivirus-mediated specific short hairpin RNA, could significantly induce cell apoptosis and inhibit the proliferative properties both in vitro and in vivo. Microarray analysis further revealed 652 upregulated genes and 527 downregulated genes in the shCCNO group compared with control, and indicated that CCNO knockdown could inhibit the malignancy of GC cells through inducing genome-wide gene expression changes.
Conclusion:
Our work is the first to reveal that elevated CCNO expression is closely associated with human GC development and that CCNO knockdown could efficiently inhibit the malignant properties of GC cells by inducing cell apoptosis. Therefore, CCNO could be used as a potential biomarker for prognosis or even as a therapeutic target in human GC.
... While S. cerevisiae only has one (Ung1), mammals have four (UNG, SMUG1, TDG, MBD4). Mammalian glyceraldehyde-3phosphate dehydrogenase (GAPDH) and cyclin O (CCNO) have also been reported to have uracil excision activity (203)(204)(205). These activities have not been characterized beyond these initial reports, though a 2015 study reported that while GAPDH binds strongly to abasic sites, no uracil excision activity was observed (206). ...
Deoxyribonucleic acid (DNA) is composed of bases that code for all functions of life. Biochemical and physical events that alter DNA include base damage and single‐ and double‐strand break (DSB) induction. Cellular DNA damage is induced through exposure to environmental chemicals and radiation, as well as from endogenous sources including normal cellular metabolism. Notably, DNA damage can occur in both the nuclear and mitochondrial genomes. Damaged DNA can lead to deleterious biological outcomes including cancer development and other diseases. Whether biological consequences arise from DNA damage largely depends on the DNA repair capacity of the cell and the interactions of the DNA damage and DNA replication machinery. Detection and measurement of cellular DNA damage is accomplished through methods that vary in sensitivity and DNA damage context.
Key Concepts
Estimated rates of induction of various deoxyribonucleic acid (DNA) lesions, including spontaneous hydrolysis and oxidative DNA damage, have been determined.
Potential biological outcomes of DNA damage include physiological conditions such as cancer, neurodegenerative diseases, heart disease and ageing.
Various exogenous and endogenous sources induce DNA base lesions and strand breaks that can be detected by various molecular and biochemical methods.
Incorporation of damaged deoxyribonucleotides during DNA synthesis can occur owing to errors committed by DNA polymerases and/or disruptions of nucleotide pool balance.
Many of the features resulting from DNA packing into chromatin, including DNA–protein interactions and sequence context, greatly affect the manner in which damage is distributed within the genome.
Cancer mutational signatures arise from context‐dependent DNA damage, and certain signatures can be attributed to specific types of DNA damage.
DNA is often the target of chemotherapeutic drugs used to treat cancer, resulting in tumour cell death.
Mitochondrial DNA damage can increase reactive oxygen species, leading to cell death, and in certain instances give rise to mutations that contribute to cancer phenotypes.
... While S. cerevisiae only has one (Ung1), mammals have four (UNG, SMUG1, TDG, MBD4). Mammalian glyceraldehyde-3phosphate dehydrogenase (GAPDH) and cyclin O (CCNO) have also been reported to have uracil excision activity (203)(204)(205). These activities have not been characterized beyond these initial reports, though a 2015 study reported that while GAPDH binds strongly to abasic sites, no uracil excision activity was observed (206). ...
DNA damage is a natural hazard of life. The most common DNA lesions are base, sugar, and single-strand break damage resulting from oxidation, alkylation, deamination, and spontaneous hydrolysis. If left unrepaired, such lesions can become fixed in the genome as permanent mutations. Thus, evolution has led to the creation of several highly conserved, partially redundant pathways to repair or mitigate the effects of DNA base damage. The biochemical mechanisms of these pathways have been well characterized and the impact of this work was recently highlighted by the selection of Tomas Lindahl, Aziz Sancar and Paul Modrich as the recipients of the 2015 Nobel Prize in Chemistry for their seminal work in defining DNA repair pathways. However, how these repair pathways are regulated and interconnected is still being elucidated. This review focuses on the classical base excision repair and strand incision pathways in eukaryotes, considering both Saccharomyces cerevisiae and humans, and extends to some important questions and challenges facing the field of DNA base damage repair.
... Depyrimidination is generally more difficult to catalyze than depurination, because pyrimidine nucleobases lack the purine N7 atom that can be protonated or otherwise activated to assist deglycosylation. 27,28 Although the uncatalyzed deglycosylation rates of purine and pyrimidine nucleosides in DNA are similar 28,29 and uracil DNA glycosylase is well known for damage repair, [30][31][32][33][34][35] natural enzymatic examples of depyrimidination of cytidine or its analogues are rare. 28,36,37 The observation of DNA-catalyzed deglycosylation at pyrimidine nucleotides further highlights the catalytic competence of DNA. ...
We report that micromolar concentrations of lanthanide ions can be required cofactors for DNA-hydrolyzing deoxyribozymes. Previous work identified deoxyribozymes that simultaneously require both Zn(2+) and Mn(2+) to achieve DNA-catalyzed DNA hydrolysis (10(12) rate enhancement); a mutant of one such DNA catalyst requires only Zn(2+). Here we show that in vitro selection in the presence of 10 µM lanthanide ion (Ce(3+), Eu(3+), or Yb(3+)) along with 1 mM Zn(2+) leads to numerous DNA-hydrolyzing deoxyribozymes that strictly require the lanthanide ion as well as Zn(2+) for catalytic activity. These DNA catalysts have a range of lanthanide dependences, including some deoxyribozymes that strongly favor one particular lanthanide ion (e.g., Ce(3+) > Eu(3+) > Yb(3+)) and others that function well with more than one lanthanide ion. Intriguingly, two of the Yb(3+)-dependent deoxyribozymes function well with Yb(3+) alone (K(d,app) ~10 µM, in the absence of Zn(2+)) and have little or no activity with Eu(3+) or Ce(3+). In contrast to these selection outcomes when lanthanide ions were present, new selections with Zn(2+) or Mn(2+) alone, or Zn(2+) with Mg(2+)/Ca(2+), led primarily to deoxyribozymes that cleave DNA by deglycosylation and β-elimination rather than by hydrolysis, including several instances of depyrimidination. We conclude that lanthanide ions warrant closer attention as cofactors when identifying new nucleic acid catalysts, especially for applications in which high concentrations of polyvalent metal ion cofactors are undesirable.
... The resulting apyrimidinic site triggers an excision-repair process (129). Two uracil-DNA glycosylases (30,77,81,86,123) and one mammalian thymine-DNA glycosylase activity have been identified in mammalian cells (83,128,129). Either of these dedicated repair pathways can be preempted by DNA replication or post-replication long-patch repair. ...
The methylation of DNA is an epigenetic modification that can play an important role in the control of gene expression in mammalian cells. The enzyme involved in this process is DNA methyltransferase, which catalyzes the transfer of a methyl group from S-adenosyl-methionine to cytosine residues to form 5-methylcytosine, a modified base that is found mostly at CpG sites in the genome. The presence of methylated CpG islands in the promoter region of genes can suppress their expression. This process may be due to the presence of 5-methylcytosine that apparently interferes with the binding of transcription factors or other DNA-binding proteins to block transcription. In different types of tumors, aberrant or accidental methylation of CpG islands in the promoter region has been observed for many cancer-related genes resulting in the silencing of their expression. How this aberrant hypermethylation takes place is not known. The genes involved include tumor suppressor genes, genes that suppress metastasis and angiogenesis, and genes that repair DNA suggesting that epigenetics plays an important role in tumorigenesis. The potent and specific inhibitor of DNA methylation, 5-aza-2′-deoxycytidine (5-AZA-CdR) has been demonstrated to reactivate the expression most of these “malignancy” suppressor genes in human tumor cell lines. These genes may be interesting targets for chemotherapy with inhibitors of DNA methylation in patients with cancer and this may help clarify the importance of this epigenetic mechanism in tumorigenesis. J. Cell. Physiol. 183:145–154, 2000. © 2000 Wiley-Liss, Inc.
... However, the misincorporation of dUTP in a RT/RNAse H assay does not appear to affect first strand DNA synthesis by RT, but rather, it affects the specificity of cleavage by RNAse H resulting in reduced second strand synthesis from the RNA primers [17]. Poxviruses also encode a UNG2 homologs that perform an essential function in the replication of this virus [22,41,43] and are thought to act at the level of DNA synthesis [8]. More recent studies confirmed that D4R is essential for vaccinia DNA synthesis, and that its essential function is unrelated to its ability to excise uracil from DNA [7]. ...
Background
Human cytomegalovirus UL114 encodes a uracil-DNA glycosylase homolog that is highly conserved in all characterized herpesviruses that infect mammals. Previous studies demonstrated that the deletion of this nonessential gene delays significantly the onset of viral DNA synthesis and results in a prolonged replication cycle. The gene product, pUL114, also appears to be important in late phase DNA synthesis presumably by introducing single stranded breaks.
Results
A series of experiments was performed to formally assign the observed phenotype to pUL114 and to characterize the function of the protein in viral replication. A cell line expressing pUL114 complemented the observed phenotype of a UL114 deletion virus in trans, confirming that the observed defects were the result of a deficiency in this gene product. Stocks of recombinant viruses without elevated levels of uracil were produced in the complementing cells; however they retained the phenotype of poor growth in normal fibroblasts suggesting that poor replication was unrelated to uracil content of input genomes. Recombinant viruses expressing epitope tagged versions of this gene demonstrated that pUL114 was expressed at early times and that it localized to viral replication compartments. This protein also coprecipitated with the DNA polymerase processivity factor, ppUL44 suggesting that these proteins associate in infected cells. This apparent interaction did not appear to require other viral proteins since ppUL44 could recruit pUL114 to the nucleus in uninfected cells. An analysis of DNA replication kinetics revealed that the initial rate of DNA synthesis and the accumulation of progeny viral genomes were significantly reduced compared to the parent virus.
Conclusion
These data suggest that pUL114 associates with ppUL44 and that it functions as part of the viral DNA replication complex to increase the efficiency of both early and late phase viral DNA synthesis.
... A cDNA highly homologous to human cyclin Og locus was reported to encode a protein with uracil-DNA glycosylase activity. 17 However, the correct sequence of both the genomic locus and several ESTs shows that this transcript does not encode the reported protein. Neither purified recombinant cyclin Oa nor cell extracts obtained after its transient transfection into HEK293 cells show detectable uracil-DNA glycosylase activity as a consequence of its overexpression as measured by an enzymatic assay 18 (data not shown). ...
We have identified an early step common to pathways activated by different forms of intrinsic apoptosis stimuli. It requires de novo synthesis of a novel cyclin, cyclin O, that forms active complexes primarily with Cdk2 upon apoptosis induction in lymphoid cells. Cyclin O expression precedes glucocorticoid and gamma-radiation-induced apoptosis in vivo in mouse thymus and spleen, and its overexpression induces caspase-dependent apoptosis in cultured cells. Knocking down the endogenous expression of cyclin O by shRNA leads to the inhibition of glucocorticoid and DNA damage-induced apoptosis due to a failure in the activation of apical caspases while leaving CD95 death receptor-mediated apoptosis intact. Our data demonstrate that apoptosis induction in lymphoid cells is one of the physiological roles of cyclin O and it does not act by perturbing a normal cellular process such as the cell cycle, the DNA damage checkpoints or transcriptional response to glucocorticoids.
... In bacterial cells, uracil-directed repair at U" G mismatches is extremely efficient due to the uracil-DNA glycosylase pathway for base excision [34]. Uracil-DNA glycosylase is found in plants [16] as well as in diverse other organisms [24,27] and is likely to be responsible for the highly (more than 9570) efficient loss of uracil residues from U" G mispairs seen in these plant transfections (Figs. 2 and 3) and in analogous mammalian cell transfections [8,14]. Such uracil excision repair had to occur before the first round of replication of the TGMV DNA sequences to explain the lack of detectable TGMV A progeny molecules with the genotype of the strand containing the U residue from the U" G mismatch. ...
We have compared the fate of U.G mispairs or analogous T.G mispairs in DNA heteroduplexes transfected into tobacco protoplasts. The heteroduplex DNA consisted of tomato golden mosaic virus DNA sequences in the Escherichia coli vectors pUC118 or pUC119. After transfection, the mismatched U residues were lost with an efficiency of greater than 95%, probably as a result of the uracil-DNA glycosylase pathway for excision of U residues in any sequence context. In contrast to the preferential removal of the mispaired U residues, biased removal of T residues from analogous heteroduplexes was not seen in the transfected plant cells. Also, we investigated the effect of extensively methylating one strand of the heteroduplex DNA used for transfection. Surprisingly, such methylation resulted in highly biased loss of the mismatched base from the 5-methylcytosine-rich strand of T.G-containing heteroduplexes.
... U:G mismatches seem to be repaired predominantly by base exci sion repair in bacterial and mammalian cells, and three UDGs have been characterized in human cells (22)(23)(24)32). In addition, other glycosylases, which excise bases with very similar structures to U such as hydroxymethyluracil-DNA glycosylase, also excise U (33). ...
Approximately 50% of mutations that inactivate the p53 tumor suppressor gene in the germline and in colon tumors are C to T transitions at methylation sites (CpG sites). These mutations are believed to be caused by an endogenous mechanism and spontaneous deamination of 5-methyl-cytosine to T is likely to contribute significantly to this high mutation rate. The resulting T:G mismatches created by this process have been hypothesized to be less efficiently repaired than U:G mismatches formed by deamination of C. We have, therefore, performed the first study to directly compare rates of T:G versus U:G base excision repair at identical sites observed to be mutated in the p53 gene using extracts of human normal colon mucosa and colon carcinoma tissue. Mismatched U was excised up to 6000-fold more efficiently than T, suggesting that differences in repair efficiencies are the major source of C to T transition mutations at CpG sites in human tissues. The data also suggests that T:G mismatches are repaired by additional mechanisms in human cells.
... Such sharing of an enzyme from a single gene has previously been demonstrated only for the yeast genes encoding photolyase (Yasui et al., 1992) and tRNA methyltransferase (Ellis et al., 1989). Two other human cDNAs possibly encoding enzymes with UDG activity have been reported (Meyer-Siegler et al., 1991;Muller and Caradonna, 1991). However, these are unrelated both to each other and to a group of homologous UDGs, and their significance is unclear, since the UNGencoded enzyme is responsible for greater than 98% of the total UDG activity in human cells (Slupphaug et al., 1995). ...
Crystal structures of the DNA repair enzyme human uracil-DNA glycosylase (UDG), combined with mutational analysis, reveal the structural basis for the specificity of the enzyme. Within the classic alpha/beta fold of UDG, sequence-conserved residues form a positively charged, active-site groove the width of duplex DNA, at the C-terminal edge of the central four-stranded parallel beta sheet. In the UDG-6-aminouracil complex, uracil binds at the base of the groove within a rigid preformed pocket that confers selectivity for uracil over other bases by shape complementary and by main chain and Asn-204 side chain hydrogen bonds. Main chain nitrogen atoms are positioned to stabilize the oxyanion intermediate generated by His-268 acting via nucleophilic attack or general base mechanisms. Specific binding of uracil flipped out from a DNA duplex provides a structural mechanism for damaged base recognition.
... It is not closely related to two other putative generate a unique pattern (Fig. 3, two bands, 10.5 and UDG encoding genes isolated from human cells (Meyer- 2.5 kb, detected with 5∞ and 3∞ probes, respectively). This Siegler et al., 1991: Müller and Caradonna, 1991). The shows that there is a single gene that hybridizes at high UDG that is most closely related is the 304 amino acid stringency to the cDNA sequence (or possibly, a number human UDG enzyme isolated and the gene cloned from of gene copies that all have the same restriction pattern human placenta ( with this enzyme). ...
Uracil-DNA glycosylase (UDG) is the enzyme responsible for the first step in the base-excision repair pathway that specifically removes uracil from DNA. Here we report the isolation of the cDNA and genomic clones for the mouse uracil-DNA glycosylase gene (ung) homologous to the major placental uracil-DNA glycosylase gene (UNG) of humans. The complete characterization of the genomic organization of the mouse uracil-DNA glycosylase gene shows that the entire mRNA coding region for the 1.83-kb cDNA of the mouse ung gene is contained in an 8.2-kb SstI genomic fragment which includes six exons and five introns. The cDNA encodes a predicted uracil-DNA glycosylase (UDG) protein of 295 amino acids (33 kDa) that is highly similar to a group of UDGs that have been isolated from a wide variety of organisms. The mouse ung gene has been mapped to mouse chromosome 5 using fluorescence in situ hybridization (FISH).
The apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide-like (APOBEC) family of single-stranded DNA (ssDNA) cytosine deaminases provides innate immunity against virus and transposon replication1–4. A well-studied mechanism is APOBEC3G restriction of human immunodeficiency virus type 1, which is counteracted by a virus-encoded degradation mechanism1–4. Accordingly, most work has focused on retroviruses with obligate ssDNA replication intermediates and it is unclear whether large double-stranded DNA (dsDNA) viruses may be similarly susceptible to restriction. Here, we show that the large dsDNA herpesvirus Epstein–Barr virus (EBV), which is the causative agent of infectious mononucleosis and multiple cancers⁵, utilizes a two-pronged approach to counteract restriction by APOBEC3B. Proteomics studies and immunoprecipitation experiments showed that the ribonucleotide reductase large subunit of EBV, BORF26,7, binds APOBEC3B. Mutagenesis mapped the interaction to the APOBEC3B catalytic domain, and biochemical studies demonstrated that BORF2 stoichiometrically inhibits APOBEC3B DNA cytosine deaminase activity. BORF2 also caused a dramatic relocalization of nuclear APOBEC3B to perinuclear bodies. On lytic reactivation, BORF2-null viruses were susceptible to APOBEC3B-mediated deamination as evidenced by lower viral titres, lower infectivity and hypermutation. The Kaposi’s sarcoma-associated herpesvirus homologue, ORF61, also bound APOBEC3B and mediated relocalization. These data support a model where the genomic integrity of human γ-herpesviruses is maintained by active neutralization of the antiviral enzyme APOBEC3B. © 2018, The Author(s), under exclusive licence to Springer Nature Limited.
The aryl hydrocarbon receptor (AhR), a transcription factor known for mediating xenobiotic toxicity, is expressed in B cells, which are known targets for environmental pollutants. However, it is unclear what the physiological functions of AhR in B cells are. We show here that expression of Ahr in B cells is up-regulated upon B-cell receptor (BCR) engagement and IL-4 treatment. Addition of a natural ligand of AhR, FICZ, induces AhR translocation to the nucleus and transcription of the AhR target gene Cyp1a1, showing that the AhR pathway is functional in B cells. AhR-deficient (Ahr(-/-)) B cells proliferate less than AhR-sufficient (Ahr(+/+)) cells following in vitro BCR stimulation and in vivo adoptive transfer models confirmed that Ahr(-/-) B cells are outcompeted by Ahr(+/+) cells. Transcriptome comparison of AhR-deficient and AhR-sufficient B cells identified cyclin O (Ccno), a direct target of AhR, as a top candidate affected by AhR deficiency.
The embryonic stem cell test (EST) is applied as a model system for detection of embryotoxicants. The application of transcriptomics allows a more detailed effect assessment compared to the morphological endpoint. Genes involved in cell differentiation, modulated by chemical exposures, may be useful as biomarkers of developmental toxicity. We describe a statistical approach to obtain a predictive gene set for toxicity potency ranking of compounds within one class. This resulted in a gene set based on differential gene expression across concentration-response series of phthalatic monoesters. We determined the concentration at which gene expression was changed at least 1.5-fold. Genes responding with the same potency ranking in vitro and in vivo embryotoxicity were selected. A leave-one-out cross-validation showed that the relative potency of each phthalate was always predicted correctly. The classical morphological 50% effect level (ID50) in EST was similar to the predicted concentration using gene set expression responses. A general down-regulation of development-related genes and up-regulation of cell-cycle related genes was observed, reminiscent of the differentiation inhibition in EST. This study illustrates the feasibility of applying dedicated gene set selections as biomarkers for developmental toxicity potency ranking on the basis of in vitro testing in the EST.
DNA repair systems are essential for the maintenance of genome integrity. Consequently, the disregulation of repair genes can be expected to be associated with significant, detrimental health effects, which can include an increased prevalence of birth defects, an enhancement of cancer risk, and an accelerated rate of aging. Although original insights into DNA repair and the genes responsible were largely derived from studies in bacteria and yeast, well over 125 genes directly involved in DNA repair have now been identified in humans, and their cDNA sequence established. These genes function in a diverse set of pathways that involve the recognition and removal of DNA lesions, tolerance to DNA damage, and protection from errors of incorporation made during DNA replication or DNA repair. Additional genes indirectly affect DNA repair, by regulating the cell cycle, ostensibly to provide an opportunity for repair or to direct the cell to apoptosis. For about 70 of the DNA repair genes listed in Table I, both the genomic DNA sequence and the cDNA sequence and chromosomal location have been elucidated. In 45 cases single-nucleotide polymorphisms have been identified and, in some cases, genetic variants have been associated with specific disorders. With the accelerating rate of gene discovery, the number of identified DNA repair genes and sequence variants is quickly rising. This report tabulates the current status of what is known about these genes. The report is limited to genes whose function is directly related to DNA repair. Environ. Mol. Mutagen. 37:241–283, 2001. © 2001 Wiley-Liss, Inc.
The Bacillus subtilis bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) protein was characterized and shown to form a stable complex with Escherichia coli uracil-DNA glycosylase (Ung). As determined by mass spectrometry, the Ugi protein had a molecular weight of 9,474. We confirmed this value by sedimentation equilibrium centrifugation and determined that Ugi exists as a monomeric protein in solution. Amino acid analysis performed on both Ugi and Ung proteins was in excellent agreement with the amino acid composition predicted from the respective nucleotide sequence of each gene. The Ung.Ugi complex was resolved from its constitutive components by nondenaturing polyacrylamide gel electrophoresis and shown to possess a 1:1 stoichiometry. Analytical ultracentrifugation studies revealed that the Ung.Ugi complex had a molecular weight of 35,400, consistent with the complex containing one molecule each of Ung and Ugi. The acidic isoelectric points of the protein species were 6.6 (Ung) and 4.2 (Ugi), whereas the Ung.Ugi complex had an isoelectric point of 4.9. Dissociation of the Ung.Ugi complex by SDS-polyacrylamide gel electrophoresis revealed no apparent alteration in the molecular weight of either polypeptide subsequent to binding. Furthermore, when the Ung.Ugi complex was treated with urea and resolved by urea-polyacrylamide gel electrophoresis, both uracil-DNA glycosylase and inhibitor activities were recovered from the dissociated complex. Thus, the complex seems to be reversible. In addition, we demonstrated that the Ugi interaction with Ung prevents enzyme binding to DNA and dissociates uracil-DNA glycosylase from a preformed DNA complex.
DNA repair mechanisms usually consist of a complex network of enzymatic reactions catalyzed by a large family of mutually interacting gene products. Thus deficiency, alteration or low levels of a single enzyme and/or of auxiliary proteins might impair a repair process. There are several indications suggesting that some enzymes involved both in DNA replication and repair are less abundant if not completely absent in stationary and non replicating cells. Postmitotic brain cell does not replicate its genome and has lower levels of several DNA repair enzymes. This could impair the DNA repair capacity and render the nervous system prone to the accumulation of DNA lesions. Some human diseases clearly characterized by a DNA repair deficiency, such as xeroderma pigmentosum, ataxia-telangiectasia and Cockayne syndrome, show neurodegeneration as one of the main clinical and pathological features. On the other hand there is evidence that some diseases characterized by primary neuronal degeneration (such as amyotrophic lateral sclerosis and Alzheimer disease) may have alterations in the DNA repair systems as well. DNA repair thus appears important to maintain the functional integrity of the nervous system and an accumulation of DNA damages in neurons as a result of impaired DNA repair mechanisms may lead to neuronal degenerations.
We have previously isolated a cDNA encoding a human uracil-DNA glycosylase which is closely related to the bacterial and yeast
enzymes. In vitro expression of this cDNA produced a protein with an apparent molecular weight of 34 K in agreement with the size predicted
from the sequence data. The in vitro expressed protein exhibited uracil-DNA glycosylase activity. The close resemblance between the human and the bacterial enzyme
raised the possibility that the human enzyme may be able to complement E. coli ung mutants. In order to test this hypothesis, the human uracil-DNA glycosylase cDNA was established in a bacterial expression
vector. Expression of the human enzyme as a LacZα-humUNG fusion protein was then studied in E. coli ung mutants. E. coli cells lacking uracil-DNA glycosylase activity exhibit a weak mutator phenotype and they are permissive for growth of phages
with uracil-containing DNA. Here we show that the expression of human uracil-DNA glycosylase in E. coli can restore the wild type phenotype of ung mutants. These results demonstrate that the evolutionary conservation of the uracil-DNA glycosylase structure is also reflected
in the conservation of the mechanism for removal of uracil from DNA.
Uracil-DNA glycosylase (UDG) is the first enzyme in the excision repair pathway for removal of uracil In DNA. In vitro transcription/translation of a cloned human cDNA encoding UDG resulted in easily measurable UDG activity. The apparent size
of the primary translation product was 34 kD. Two lines of evidence indicated that this cDNA encodes the major nuclear UDG.
First, In vitro translation of human fibroblast mRNA isolated from S-phase cells resulted In measurable UDG activity and this UDG translation
was specifically inhibited 90% by an anti-sense UDG mRNA transcript. Secondly, cell cycle analysis revealed an 8–12 fold Increase
in transcript level late in the G1-phase preceding a 2 – 3 fold increase in total UDG activity in the S-phase. UDG degradation was found to be very slow (T1/2 = 30h), therefore, the rate of UDG synthesis could be derived from the rate of UDG accumulation, and was found to correlate
temporarily and quantitatively with the transcript level. Inhibitor studies showed that RNA and protein synthesis was required
for induction of UDG. However, specific inhibition of DNA replication with aphldlcolin indicated that entrance of fibroblasts
into the S-phase was not required for UDG accumulation.
The cell cycle-dependent transcription factor, E2F-1, regulates the cyclin-like species of the DNA repair enzyme uracil-DNA glycosylase (UDG) gene in human osteosarcoma (Saos-2) cells. We demonstrate, through the deletion of the human UDG promoter sequences, that expression of E2F-1 activates the UDG promoter through several E2F sites. The major putative downstream site for E2F, located in the first exon, serves as a target for E2F-1/DP1 complex binding in vitro. We also provide evidence for the functional relationship between the cyclin-like UDG gene product and E2F. High levels of UDG expression in a transient transfection assay result in the down-regulation of transcriptional activity through elements specific for E2F-mediated transcription. Overexpression of UDG in Saos 2 cells was observed to delay growth late in G1 phase and transiently arrest these cells from progressing into the S phase. This hypothetical model integrates one mechanism of DNA repair with the cell cycle control of gene transcription, likely through E2F. This implicates E2F as a multifunctional target for proteins and enzymes, possibly, responsive to DNA damage through the negative effect of UDG on E2F-mediated transcriptional activity.
The developmental regulation of the mammalian DNA-repair enzyme uracil DNA glycosylase was examined in the rat at specific intervals ranging from -4 days before to 106 days after birth. Enzyme activity was quantitated by in vitro biochemical assay. In the adult animal, as measured in crude cell extracts, three organs (liver, kidney and spleen) had significant levels of activity. In contrast, three organs (brain, heart and lung) had low activity. Partial purification of this enzyme identified one major species of molecular weight 32,700 Da, demonstrating the quantitation of the nuclear glycosylase. During development, with the exception of the liver, the specific activity of the glycosylase paralleled the regulation of DNA synthesis. In these organs the highest levels of the glycosylase and the rate of DNA replication were observed around the time of birth. In the liver, DNA replication was similarly regulated. However, glycosylase activity was minimal at early stages of life. Instead, maximal levels were observed at 14-21 days after birth. At that time DNA replication was severely reduced. These results demonstrate that individual organs express this DNA-repair enzyme in a distinct and specific pattern during development. Accordingly, the regulation of the uracil DNA glycosylase during development may provide a model system to examine the differential regulation of DNA-repair genes.
Uracil residues are introduced into prokaryotic and eukaryotic deoxyribonucleic acid (DNA) as a normal physiological process during DNA synthesis, and by spontaneous chemical modification of cytosine residues in DNA; thus, the acquisition of uracil in cellular DNA is unavoidable. However, the rate of uracil accumulation may vary significantly, depending on the ratio of deoxyuridine triphosphate (dUTP) to deoxythymidine triphosphate (dTTP) in intracellular pools and on whether the cells are exposed to cytosinedeaminating agents. The biological consequences of uracil residues in DNA may have cytotoxic, mutagenic, or lethal effects. An uncontrolled accumulation of the uracil residues in DNA leads to various perturbations of molecular events, ranging from altered protein-nucleic acid interactions to uracil-DNA degradation. The importance of eliminating uracil from DNA is underscored, by the observation that the uracil-DNA repair pathway of almost every organism examined, is remarkably similar. It appears that not only is one nucleotide DNA repair evident in E. coli as well as in human cells, but also that uracil-DNA glycosylase is one of the most highly conserved polypeptides yet identified.
A human small cell lung cancer cell line, U-1906, developed altered functional properties upon continuous in vitro cultivation. Cells obtained at late (U-1906 L) and early (U-1906 E) passages of cultivation differ in drug resistance to the cytostatic therapeutic agents cisplatin and doxorubicin. The U-1906 L cells are 1.6-fold and 1.3-fold more resistant to cisplatin and doxorubicin respectively, than are the U-1906 E cells. In the more resistant U-1906 L cells, the total glutathione (GSH plus GSSG) level is 40% lower, whereas the activities of GSH-linked enzymes such as GSH peroxidase and GSH transferases are significantly higher. Quantitative analysis with isoenzyme-specific ELISAs demonstrated increased concentrations of all three of the measurable GSTs, M1-1, M3-3 and P1-1, in the more resistant cells. The intracellular protein expression patterns of the U-1906 E and the U-1906 L cells are very similar as revealed by two-dimensional denaturing electrophoresis, but show significant alterations in the concentrations of some components. Two 35 kDa proteins of different pI values, the concentrations of which are increased in the U-1906 L cells, were both identified as glyceraldehyde-3-phosphate dehydrogenase, either by N-terminal or by internal amino acid sequence analysis. The present study demonstrates that the increased resistance of the U-1906 L cells may involve multiple detoxification mechanisms and that the contribution of the GSH-linked detoxification can be ascribed to the elevation of cytosolic GST isoenzymes, GSH peroxidase and glutathione reductase, rather than to the intracellular GSH concentrations.
Photochemical cross-linking of Escherichia coli uracil-DNA glycosylase (Ung) to oligonucleotide dT20 was performed to identify amino acid residues that reside in or near the DNA-binding site. UV-catalyzed cross-linking reactions produced a covalent Ung x dT20 complex which was resolved from uncross-linked enzyme by SDS-polyacrylamide gel electrophoresis. Cross-link formation required native Ung and was inhibited by increasing concentrations of NaCl in a manner characteristics of NaCl inhibition of Ung catalytic activity. The Ung x dT20 complex was purified to apparent homogeneity, and mass spectrometry revealed that Ung was cross-linked to dT20 in 1:1 stoichiometry as a 31,477 dalton complex. Purified Ung x dT20 lacked detectable uracil-DNA glycosylase activity and failed to bind single-stranded DNA. Recently, we demonstrated that the bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) binds Ung and prevents further interaction with DNA (Bennett, S. E., Schimerlik, M. I., and Mosbaugh, D. W. (1993) J. Biol. Chem. 268, 26879-26885). Addition of the Ugi protein to the cross-linking reaction blocked formation of the Ung x dT20 cross-link. Conversely, the Ung x dT20 cross-link was refractory to Ugi binding. Upon trypsin digestion of Ung x dT20, four distinct products were identified as peptide x dT20 cross-links. A combination of amino acid sequence and mass spectrometric analysis revealed that four tryptic peptides (T6, T18, T19, and T18/19) were adducted to dT20. These observations suggest that dT20 is cross-linked to the Ung DNA-binding site.
Recent cloning of a cDNA (UNG15) encoding human uracil-DNA glycosylase (UDG), indicated that the gene product of Mr = 33,800 contains an N-terminal sequence of 77 amino acids not present in the presumed mature form of Mr= 25,800. This led to the hypothesis that the N-termlnal sequence might be involved in intracellular targeting. To examine
this hypothesis, we analysed UDG from nuclei, mitochondria and cytosol by western blotting and high resolution gel filtration.
An antibody that recognises a sequence in the mature form of the UNG protein detected all three forms, indicating that they
are products of the same gene. The nuclear and mitochondrial form had an apparent Mr= 27,500 and the cytosolic form an apparent Mr= 38,000 by western blotting. Gel filtration gave essentially similar estimates. An antibody with specificity towards the
presequence recognised the cytosolic form of Mr= 38,000 only, indicating that the difference in size is due to the presequence. Immunofluorescence studies of HeLa cells
clearly demonstrated that the major part of the UDG activity was localised in the nuclei. Transfection experiments with plasmids
carrying full-length UNG15 cDNA or a truncated form of UNG15 encoding the presumed mature UNG protein demonstrated that the UNG presequence mediated sorting to the mitochondria, whereas
UNG lacking the presequence was translocated to the nuclei. We conclude that the same gene encodes nuclear and mitochondrial
uracil-DNA glycosylase and that the signals for mitochondrial translocation resides in the presequence, whereas signals for
nuclear import are within the mature protein.
An open reading frame, BamHI D6R, from the central highly conserved region of the Shope fibroma virus (SFV) genome was sequenced and found to have significant homology to that of uracil DNA glycosylases from a number of organisms. Uracil DNA glycosylase catalyzes the initial step in the repair pathway that removes potentially mutagenic uracil from duplex DNA. The D6R polypeptide was expressed in reticulocyte lysates programmed with RNA transcribed from an expression vector containing the T7 RNA polymerase promoter. A highly specific ethidium bromide fluorescence assay of the in vitro translation product determined that the encoded protein does indeed possess uracil DNA glycosylase activity. Open reading frames from other poxviruses, including vaccinia virus (HindIII D4R) and fowlpox (D4), are highly homologous to D6R of SFV and are predicted to encode uracil DNA glycosylases. Identification of the SFV uracil DNA glycosylase provides evidence that this poxviral protein is involved in the repair of the viral DNA genome. Since this enzyme performs only the initial step required for the removal of uracil from DNA, creating an apyrimidinic site, we suggest that other, possibly virus-encoded, repair activities must be present in the cytoplasm of infected cells to complete the uracil excision repair pathway.
The herpes simplex virus type 2 (HSV-2) uracil-DNA glycosylase (UNG) was from the nuclei of infected KB cells using ion exchange, affinity, and chromatofocusing chromatography techniques. Chromatography on DNA cellulose revealed two distinct HSV-2-encoded UNGs. One species, designated A, was purified approximately 324-fold, while the second species, designated B, was purified approximately 130-fold. The HSV UNG species B was observed to unidirectionally convert to the A species, suggesting that the B species binding to DNA cellulose may be the result of an association with other DNA binding proteins. SDS-PAGE demonstrated that both species A and B had molecular weights of 32,000. The HSV-2-encoded UNGs could be distinguished from the cellular nuclear UNG based upon differences in their behavior on the chromatography matrixes and by their molecular weights. There were no significant differences in the biochemical properties of the HSV-2-encoded or nuclear UNGs. Furthermore, all of these UNGs reacted with a monoclonal antibody produced against the human placental UNG. These data support recent studies, at both the DNA and the amino acid levels, which have demonstrated that this enzyme is highly conserved between species.
We have determined the nucleotide sequence and transcription pattern of a group of open reading frames of pseudorabies virus (PRV), which are located at the right end of the BamHI-G fragment from 0.664 to 0.695 map units in the unique long region of the genome. Nucleotide sequence analysis revealed four open reading frames. The first three correspond in genome location to the herpes simplex virus type 1 (HSV-1) open reading frames UL1, which codes for glycoprotein L (gL); UL2, which codes for a uracil-DNA glycosylase; and UL3, which codes for a polypeptide of unknown function. The fourth open reading frame, UL3.5, is not present in the HSV-1 genome. Northern (RNA) blot analysis with oligonucleotide and cDNA probes revealed four abundant mRNA species of 3.3, 2.7, 1.8, and 0.9 kb, which are likely to yield polypeptides encoded by the UL1, -2, -3, and -3.5 open reading frames, respectively. All four transcripts were of the early-late kinetic class, transcribed in the same direction, and 3' coterminal. The UL2 and UL3 genes of PRV and HSV-1 have significant amino acid sequence homology, while the UL1 genes are positional homologs and the UL3.5 gene is unique to PRV.
The predicted amino acid sequence of a human cDNA encoding uracil-DNA glycosylase activity shows striking similarity to the cyclin protein family. To characterize the expression of this DNA repair enzyme, we have isolated the corresponding genomic clone. This gene is contained within 4.2 kilobases and is composed of only two exons. Sequence analysis of the upstream region shows that it contains two cell cycle box (CCB) regulatory elements which are also found in yeast cyclin genes. Deletional analysis of the promoter reveals the presence of a repressor region located from position -812 to -603. An inverted CCB element (alpha-CCB) and an SP1-like binding site are contained within this region. When uracil-DNA glycosylase mRNA levels are examined in vivo, a 2-3-fold increase is associated with G1 phase in both HeLa S3 and WI38 cells. To examine the role of the 209-base pair repressor region in mediating cell cycle regulation, this fragment was used in gel shift assays with cellular extracts prepared from various stages of the cell cycle. Several specific complexes are formed during S and G2 phases which are not present during M and G1 phases. Two of the complexes are the result of alpha-CCB binding as they can be specifically disrupted by the addition of an oligonucleotide containing the alpha-CCB binding site. Immunoprecipitation studies reveal that uracil-DNA glycosylase protein levels are also elevated during G1 phase. Additionally, we show that the 36-kDa uracil-DNA glycosylase protein is turned over during the course of one cell cycle. These results demonstrate coordinate regulation of uracil-DNA glycosylase at both the transcriptional and the post-transcriptional levels as a function of the cell cycle.
We have purified uracil DNA-glycosylase (UDG) from calf thymus 32,OOO-fold and studied Its biochemical properties, including
sequence specificity. The enzyme is apparently closely related to human UDG, since it was recognised by a polyclonal antibody
directed towards human UDG. SDS-PAGE and western analysis Indicate an apparent Mr = 27,500. Bovine UDG has a 1.7-fold preference
for single stranded over double stranded DNA as a substrate. Sequence specificity for uracil removal from dsDNA was examined
for bovine and Escherichla coll UDG, using DNA containing less than one dUMP residper 100 nucleotides and synthetic oligonucleotides containing one dUMP
residue. Comparative studies Involving about 40 uracil sites Indicated similar specificities for both UDGs. We found more
than a 10-fold difference In rates of uracil removal between different sequences. 5’-G/cUT-3’ and 5’-G/cUG/c-3’ were consensus sequences for poor repair whereas 5’-A/TUAA/T-3’ was a consensus for good repair. Sequence specificity was verified in double stranded oligonucleotides, but not in single
stranded ones, suggesting that the structure of the double stranded DNA helix has Influence on sequence specificity. Rate
of uracil removal appeared to be slightly faster from U:A base pairs as compared to U:G mis-matches. The results indicate
that sequence specific repair may be a determinant to be considered in mutagenesis.
Evidence is presented on two forms of uracil-DNA glycosylase (UDG1 and UDG2) that exist in human cells. We have developed an affinity technique to isolate uracil-DNA glycosylases from HeLa cells. This technique relies on the use of a uracil-DNA glycosylase inhibitor (Ugi) produced by the Bacillus subtilis bacteriophage, PBS2. Affinity-purified preparations of uracil-DNA glycosylase, derived from total HeLa cell extracts, reveal a group of bands in the 36,000 molecular weight range and a single 30,000 molecular weight band when analyzed by SDS-PAGE and silver staining. In contrast, only the 30,000 molecular weight band is seen in HeLa mitochondrial preparations. Separation of HeLa cell nuclei from the postnuclear supernatant reveals that uracil-DNA glycosylase activity is evenly distributed between the nuclear compartment and the postnuclear components of the cell. Immunostaining of a nuclear extract with antisera to UDG1 indicates that the nuclear associated uracil-DNA glycosylase activity is not associated with the highly conserved uracil-DNA glycosylase, UDG1. With the use of Ugi-Sepharose affinity chromatography, we show that a second and distinct uracil-DNA glycosylase is associated with the nuclear compartment. Immunoblot analysis, utilizing antisera generated against UDG1, reveals that the 30,000 molecular weight protein and a protein in the 36,000 range share common epitopes. Cycloheximide treatment of HeLa cells indicates that upon inhibition of protein synthesis, the higher molecular weight species disappears and is apparently post-translationally processed into a lower molecular weight form. This is substantiated by mitochondrial import studies which reveal that in vitro expressed UDG1 becomes resistant to trypsin treatment within 15 min of incubation with mitochondria. Within this time frame, a lower molecular weight form of uracil-DNA glycosylase appears and is associated with the mitochondria. Antibodies generated against peptides from specific regions of the cyclin-like uracil-DNA glycosylase (UDG2), demonstrate that this nuclear glycosylase is a phosphoprotein with a molecular weight in the range of 36,000. SDS-PAGE analysis of Ugi affinity-purified and immunoprecipitated UDG2 reveals two closely migrating phosphate-containing species, indicating that UDG2 either contains multiple phosphorylation sites (resulting in heterogeneous migration) or that two distinct forms of UDG2 exist in the cell. Cell staining of various cultured human cell lines corroborates the finding that UDG1 is largely excluded from the nucleus and that UDG2 resides mainly in the nucleus. Our results indicate that UDG1 is targeted to the mitochondria and undergoes proteolytic processing typical of resident mitochondrial proteins that are encoded by nuclear DNA. These results also indicate that the cyclin-like uracil-DNA glycosylase (UDG2) may be a likely candidate for the nuclear located base-excision repair enzyme.
Human cytomegalovirus (CMV) encodes a gene, UL114, whose product is homologous to the uracil DNA glycosylase and is highly conserved in all herpesviruses. This DNA repair enzyme excises uracil residues in DNA that result from the misincorporation of dUTP or spontaneous deamination of cytosine. We constructed a recombinant virus, RC2620, that contains a large deletion in the UL114 open reading frame and carries a 1.2-kb insert containing the Escherichia coli gpt gene. RC2620 retains the capacity to replicate in primary human fibroblasts and reaches titers that are similar to those produced by the parent virus but exhibits a significantly longer replication cycle. Although the rate of expression of alpha and beta gene products appears to be unaffected by the mutation, DNA synthesis fails to proceed normally. Once initiated, DNA synthesis in mutant virus-infected cells proceeds at the same rate as with wild-type virus, but initiation is delayed by 48 h. The mutant virus also exhibits two predicted phenotypes: (i) hypersensitivity to the nucleoside analog 5-bromodeoxyuridine and (ii) retention of more uracil residues in genomic DNA than the parental virus. Together, these data suggest UL114 is required for the proper excision of uracil residues from viral DNA but in addition plays some role in establishing the correct temporal progression of DNA synthesis and viral replication. Although such involvement has not been previously observed in herpesviruses, a requirement for uracil DNA glycosylase in DNA replication has been observed in poxviruses.
We have previously isolated a human gene that encodes a cyclin-like protein with uracil-removing activity (UDG2) (Muller, S.J., and Caradonna, S. 1993. J. Biol. Chem. 268, 1310-1319). The structural and regulatory similarities shared between this uracil-DNA glycosylase and cyclins suggested that it may interact with additional proteins. Using a unique affinity purification protocol (Ugi-Sepharose) and anti-UDG2 antibodies, we have identified a physical interaction between the cyclin-like uracil-DNA glycosylase and PCNA in extracts derived from HeLa cells. Conversely, we show that anti-PCNA immunoprecipitates possess significant uracil-DNA glycosylase activity. This activity is specifically blocked by the addition of uracil-DNA glycosylase inhibitor protein (Ugi) derived from bacteriophage PBS2. To further characterize this association, we performed in vitro mixing experiments using 35S-labeled PCNA and uracil-DNA glycosylase (UDG2) that were generated in a coupled transcription/translation system. We show that UDG2 and PCNA are coprecipitated using anti-PCNA antibodies and anti-UDG2 antibodies as well as Ugi-Sepharose. When PCNA is preincubated with synthetic peptides corresponding to amino acid residues 73-90 of UDG2, the PCNA-UDG2 association is prevented. By contrast, addition of synthetic peptides corresponding to amino acid residues 208-223 has no effect on this interaction. These findings suggest that the UDG2 domain encompassing amino acids 73-90 is directly involved in binding PCNA.
The human uracil-DNA glycosylase gene (UNG) spans approximately 13.5 kb including the promoter. UNG comprises 6 exons and 5 introns and was assigned to chromosome 12q23-q24.1 by radiation hybrid mapping. UNG exhibits typical features of housekeeping genes, including a 5' CpG island of 1.2 kb and a very GC-rich TATA-less promoter containing a number of elements involved in constitutive expression and cell cycle regulation. A smaller CpG island is located just downstream of the gene. Within the 15-kb sequence we identified 16 Alu retroposons, 2 of which contain putative competent RNA polymerase III promoters, 3 copies of medium reiteration frequency repeats, and 1 copy of a mammalian-wide interspersed repetitive element, as well as a 300-bp TA-dinucleotide repeat. In vitro methylation of the UNG promoter strongly reduced promoter activity, but methylation may not be involved in regulation of UNG in vivo since a narrow region of the 5' CpG island comprising the putative transcription factor binding region appears to be invariably methylation-free.
A distinct nuclear form of human uracil-DNA glycosylase [UNG2, open reading frame (ORF) 313 amino acid residues] from the
UNG gene has been identified. UNG2 differs from the previously known form (UNG1, ORF 304 amino acid residues) in the 44 amino
acids of the N-terminal sequence, which is not necessary for catalytic activity. The rest of the sequence and the catalytic
domain, altogether 269 amino acids, are identical. The alternative N-terminal sequence in UNG2 arises by splicing of a previously
unrecognized exon (exon 1A) into a consensus splice site after codon 35 in exon 1B (previously designated exon 1). The UNG1
sequence starts at codon 1 in exon 1B and thus has 35 amino acids not present in UNG2. Coupled transcription/translation in
rabbit reticulocyte lysates demonstrated that both proteins are catalytically active. Similar forms of UNG1 and UNG2 are expressed
in mouse which has an identical organization of the homologous gene. Constructs that express fusion products of UNG1 or UNG2
and green fluorescent protein (EGFP) were used to study the significance of the N-terminal sequences in UNG1 and UNG2 for
subcellular targeting. After transient transfection of HeLa cells, the pUNG1-EGFP-N1 product colocalizes with mitochondria,
whereas the pUNG2-EGFP-N1 product is targeted exclusively to nuclei.
Excision of uracil from tetraloop hairpins and single stranded ('unstructured') oligodeoxyribonucleotides by Escherichia coli uracil DNA glycosylase has been investigated. We show that, compared with a single stranded reference substrate, uracil from the first, second, third and the fourth positions of the loops is excised with highly variable efficiencies of 3.21, 0.37, 5.9 and 66.8%, respectively. More importantly, inclusion of E.coli single stranded DNA binding protein (SSB) in the reactions resulted in approximately 7-140-fold increase in the efficiency of uracil excision from the first, second or the third position in the loop but showed no significant effect on its excision from the fourth position. In contrast, the presence of SSB decreased uracil excision from the single stranded ('unstructured') substrates approximately 2-3-fold. The kinetic studies show that the increased efficiency of uracil release from the first, second and the third positions of the tetraloops is due to a combination of both the improved substrate binding and a large increase in the catalytic rates. On the other hand, the decreased efficiency of uracil release from the single stranded substrates ('unstructured') is mostly due to the lowering of the catalytic rates. Chemical probing with KMnO4showed that the presence of SSB resulted in the reduction of cleavage of the nucleotides in the vicinity of dUMP residue in single stranded substrates but their increased susceptibility in the hairpin substrates. We discuss these results to propose that excision of uracil from DNA-SSB complexes by uracil DNA glycosylase involves base flipping. The use of SSB in the various applications of uracil DNA glycosylase is also discussed.
Base excision repair is a major mechanism for correcting aberrant DNA bases. We are using an in vitro base excision repair assay to fractionate and purify proteins from a human cell extract that are involved in this type of repair. Three fractions are required to reconstitute base excision repair synthesis using a uracil-containing DNA as a model substrate. We previously showed that one fraction corresponds to DNA polymerase beta. A second fraction was extensively purified and found to possess uracil-DNA glycosylase activity and was identified as the product of the UNG gene. A neutralizing antibody to the human UNG protein inhibited base excision repair in crude extract by at least 90%. The third fraction was highly purified and exhibited apurinic/apyrimidinic (AP) endonuclease activity. Immunoblot analysis identified HAP1 as the major polypeptide in fractions possessing DNA repair activity. Recombinant versions of UNG, HAP1, and DNA polymerase beta were able to substitute for the proteins purified from human cells. Addition of DNA ligase I led to ligated repair products. Thus, complete base excision repair of uracil-containing DNA was achieved by a combination of UNG, HAP1, DNA polymerase beta, and DNA ligase I. This is the first complete reconstitution of base excision repair using entirely eukaryotic proteins.
Deamination of cytosine residues contributes to the appearance of uracil in DNA. Uracil DNA glycosylase (UDG) initiates uracil excision repair to safeguard the genomic integrity. To study the mechanism of uracil excision in mycobacteria (organisms with G+C rich genomes), we have purified UDG from Mycobacterium smegmatis by more than 3000-fold. The molecular mass of M. smegmatis UDG, as determined by SDS/PAGE, is approximately 25 kDa and it shows maximum activity at pH 8.0. The N-terminal sequence analysis shows that the initiating amino acid, formyl-methionine is cleaved from the mature protein. More interestingly, unlike Escherichia coli UDG, which forms a physiologically irreversible complex with the inhibitor protein Ugi, M. smegmatis UDG forms a dissociable complex with it. M. smegmatis UDG excises uracil from the 5'-terminal position of the 5'-phosphorylated substrates. However, its excision from the 3'-penultimate position is extremely poor. Similar to E. coli UDG, M. smegmatis UDG also uses pd(UN)p as its minimal substrate. However, in contrast to E. coli UDG, which excises uracil from different loop positions of tetraloop hairpin substrates with highly variable efficiencies, M. smegmatis UDG excises the same uracil residues with comparable efficiencies. Kinetic parameters (Km and Vmax) for uracil release from synthetic substrates suggest that M. smegmatis UDG is an efficient enzyme and better suited for molecular biology applications. We discuss the usefulness of the distinct biochemical properties of M. smegmatis UDG in the possible design of selective inhibitors against it.
NAD(P)H:quinone oxidoreductase (NQO1; DT-diaphorase) is elevated in certain tumors, such as non-small cell lung cancer (NSCLC). Compounds such as mitomycin C and streptonigrin are efficiently bioactivated by NQO1 and have been used in an enzyme-directed approach to chemotherapy. Previously, 2,5-diaziridinyl-3,6-dimethyl-1,4-benzoquinone (MeDZQ) was identified as a potential antitumor agent based on its high rate of bioactivation by human NQO1 and its selective cytotoxicity to cells containing elevated NQO1. RH1, a water-soluble analogue of MeDZQ synthesized in this work, was a better substrate for recombinant human NQO1 than the parent compound. RH1 was, correspondingly, more cytotoxic to human tumor cells expressing elevated NQO1 activity (H460 NSCLC and HT29 human colon carcinoma), as measured by 3-(4,5-dimethylthiazol-2,5-diphenyl)tetrazolium assay, than it was to cells deficient in NQO1 activity (H596 NSCLC and BE human colon carcinoma). RH1 exhibited a greater selective toxicity (ratio of IC50s in H596:H460 and BE:HT29) to cells with elevated NQO1 activity relative to MeDZQ. Additionally, we report the establishment of a stable line of BE human colon carcinoma cells transfected with wild-type human NQO1 (BE-NQ7). BE cells are devoid of NQO1 activity due to a homozygous point mutation in the NQO1 gene. In comparison to the parental cell line, RH1, MeDZQ, and mitomycin C were significantly more cytotoxic to BE-NQ7 cells (17-, 7-, and 3-fold, respectively), confirming that the presence of NQO1 is sufficient to increase cytotoxicity of these antitumor quinones. These data suggest that RH1 may be an effective NQO1-directed antitumor agent for the therapy of tumors with elevated NQO1 activity, such as NSCLC.
Multiple genetic alterations are commonly observed in human cancers. It has been suggested that inactivation of DNA repair pathways, which leads to an increased mutation rate and chromosomal instability, can initiate and accelerate the neoplastic process. Such a causality has been shown for DNA mismatch repair and Hereditary Nonpolyposis Colorectal Cancer (HNPCC), and evidence is accumulating that several other DNA repair pathways are frequently inactivated in different cancer types. In addition to genetic alterations, perturbations in DNA methylation patterns (epigenetic changes), which include both local hypermethylation and genome-wide hypomethylation, are frequently observed early in tumorigenesis. Therefore, genomic instability including genetic and/or epigenetic alterations may be the first step in carcinogenesis. Knowledge of these biochemical mechanisms are likely to lead to more effective cancer diagnosis and therapy.
It has been reported that Drosophila lacks a uracil-DNA glycosylase but that a direct incising activity on uracil-containing DNA appeared developmentally only in third instar larvae. In contrast we have found by two independent assays, that uracil-DNA glycosylase exists in both Drosophila eggs as well as in third instar larvae. The first assay shows the liberation of [³H] uracil from a d(AT)n polymer randomly substituted with [³H]uracil by its synthesis in the presence of [³H] dUTP. The second fluorometric assay for uracil-DNA glycosylase depends on the unique topological properties of circular DNAs and has the advantage of detecting apyrimidinic/apurinic (AP) endonuclease activity as well. To test one other insect, locust eggs were also assayed for uracil-DNA glycosylase. The amount of uracil-DNA glycosylase correlated well with the amount of DNA in actively replicating cells.
Uracil-DNA glycosylase is the DNA repair enzyme responsible for the removal of uracil from DNA, and it is present in all organisms investigated. Here we report on the cloning and sequencing of a cDNA encoding the human uracil-DNA glycosylase. The sequences of uracil-DNA glycosylases from yeast, Escherichia coli, herpes simplex virus type 1 and 2, and homologous genes from varicella-zoster and Epstein-Barr viruses are known. It is shown in this report that the predicted amino acid sequence of the human uracil-DNA glycosylase shows a striking similarity to the other uracil-DNA glycosylases, ranging from 40.3 to 55.7% identical residues. The proteins of human and bacterial origin were unexpectedly found to be most closely related, 73.3% similarity when conservative amino acid substitutions were included. The similarity between the different uracil-DNA glycosylase genes is confined to several discrete boxes. These findings strongly indicate that uracil-DNA glycosylases from phylogenetically distant species are highly conserved.
The structural gene for the Saccharomyces cerevisiae repair enzyme uracil-DNA-glycosylase (UNG1) was selected from a yeast genomic library in the multicopy vector YEp24 by complementation of the ung1-1 mutant in in vitro enzyme assays. The sequenced gene has an open reading frame which codes for a protein with molecular weight of 40,471. The measured size of the mRNA of 1.25 kb is in agreement with the predicted molecular weight of the protein. The gene product was overproduced about 100-fold in strains carrying an UNG1 gene containing plasmid at 100-200 copies/cell. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cleared lysates from such an overproducing strain, followed by renaturation of enzyme activity from individual gel slices showed the presence of two enzymatic activities in comparable quantities with Mr values of 39,500 and 33,000, indicating that the full size protein is either readily degraded in vivo or is very sensitive to proteolytic digestion in vitro. The carboxyl-terminal two-thirds of the yeast uracil-DNA-glycosylase is highly homologous to the entire Escherichia coli enzyme (50% amino acid identity). Genetic mapping experiments have localized the UNG1 gene on the left arm of chromosome XIII at 17 cM from the GAL80 locus proximal to the centromer. Deletions of the UNG1 gene are viable.
A series of anti-human placental uracil DNA glycosylase monoclonal antibodies was used to screen a human placental cDNA library in phage lambda gt11. Twenty-seven immunopositive plaques were detected and purified. One clone containing a 1.2-kilobase (kb) human cDNA insert was chosen for further study by insertion into pUC8. The resultant recombinant plasmid selected by hybridization a human placental mRNA that encoded a 37-kDa polypeptide. This protein was immunoprecipitated specifically by an anti-human placental uracil DNA glycosylase monoclonal antibody. RNA blot-hybridization (Northern) analysis using placental poly(A)+ RNA or total RNA from four different human fibroblast cell strains revealed a single 1.6-kb transcript. Genomic blots using DNA from each cell strain digested with either EcoRI or Pst I revealed a complex pattern of cDNA-hybridizing restriction fragments. The genomic analysis for each enzyme was highly similar in all four human cell strains. In contrast, a single band was observed when genomic analysis was performed with the identical DNA digests with an actin gene probe. During cell proliferation there was an increase in the level of glycosylase mRNA that paralleled the increase in uracil DNA glycosylase enzyme activity. The isolation of the human uracil DNA glycosylase gene permits an examination of the structure, organization, and expression of a human DNA repair gene.
The complete nucleotide sequence of the Escherichia coli ung gene is described. Transcription initiation and termination sites were determined by S1 nuclease and RNase mapping. The common prokaryotic -35, -10, and the ribosome binding site sequences are represented by TGTTCTGTA, TAAGCTA, and AGGAGAG at their respective locations. A putative hairpin transcription terminator structure is present at the major transcription terminator sites. The open reading frame of the ung gene codes for a protein of 229 amino acids (25,664 daltons). The molecular weight, amino acid composition, and the N-terminal amino acid sequence of the uracil DNA glycosylase purified from E. coli cells match with the open reading frame of the ung gene. The protein sequence analysis shows that the N-terminal methionine is cleaved off in the mature protein. The in vitro transcription coupled translation of the ung gene directs the synthesis of a protein which comigrates with uracil DNA glycosylase. Also, the CNBr cleavage of the protein synthesized in vitro confirms the positions of the methionines deduced from the DNA sequence. The levels of ung gene expression remain constant up to the early stationary phase, but decline in the late stationary phase of the E. coli culture. The E. coli gene showed a strong sequence homology to Shigella, a weak sequence homology to Salmonella and Citrobacter, and a very weak sequence homology to Proteus genes. No sequence homologies were seen for Pseudomonas, Clostridium, Micrococcus, and several eukaryotic genomes.
We have recently isolated a herpes simplex virus (HSV) type 2 (strain 333)-specific cDNA that encodes uracil-DNA glycosylase. This cDNA lies between 0.065 and 0.08 map units on the HSV genome. Within this region there are five overlapping transcripts which encompass three open reading frames. We have determined that the second open reading frame, UL-2, codes for glycosylase. In vitro transcription of the UL-2 region and subsequent translation yielded uracil-DNA glycosylase activity. Sequence analysis of the UL-2 open reading frame indicated a coding capacity of 295 amino acids. Comparison to the HSV type 1 (strain 17) sequence indicated that there is 74% amino acid homology between the two strains, with most of the conservation occurring in the middle and the 3' end. The 5' end, however, has diverged considerably.
PA101 and PA104 are Rous sarcoma virus variants that are differentially temperature sensitive in cell transformation parameters, including stimulation of cell proliferation, morphological alteration, and anchorage independence. To investigate the biochemical basis for the differential expression of these parameters, the tyrosine kinase activity and subcellular localization of the mutant p60v-src proteins encoded in the variants were examined. Analysis of chimeric src proteins derived from the mutant proteins revealed that lesions in the kinase domain inhibit in vitro kinase activity and confer temperature sensitivity on tyrosine phosphorylation of cellular protein p34 in vivo. The amino-terminal portions of the mutant src proteins also influence tyrosine phosphorylation in vivo and in vitro, which is consistent with an interaction between an amino-terminal region and the kinase domain. Large proportions of the mutant src proteins exist in soluble complexes with cellular proteins p50 and p90, even though the src proteins are myristylated. The formation of these soluble complexes segregates with lesions in the kinase domain and is independent of temperature. Our results demonstrate that the transformation parameters examined correlate to a limited extent with p34 phosphorylation but not with the levels of in vitro kinase activity or soluble complex formation.
Activity of the DNA repair enzyme uracil-DNA glycosylase has been shown to increase in herpes simplex virus type 2 (HSV-2)-infected cells. When mRNA derived from either HSV-1- or HSV-2-infected HeLa S3 cells was translated in an in vitro translation system, significant uracil-DNA glycosylase activity could be detected in the lysate. This activity was specific for the removal of uracil from DNA. Lysates from in vitro translation of mRNA derived from uninfected HeLa cells did not contain measurable glycosylase activity. A cDNA library was constructed with mRNA derived from HSV-2-infected cells 10 h postinfection. Pooled isolates from this library were used in hybrid-arrest and in vitro translation reactions to isolate a uracil-DNA glycosylase-specific cDNA. In vitro translation of hybrid-selected RNA, by using this cDNA, produced glycosylase activity in the lysate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radiolabeled products from this translation reaction showed a protein component with a molecular weight of 39,000. This is consistent with the molecular weight determinations of the purified glycosylase enzyme derived from either uninfected or HSV-infected HeLa cells. Northern (RNA blot) analysis of HSV-derived RNA, by using the glycosylase cDNA as a probe, revealed five overlapping transcripts of 3.4, 2.8, 2.4, 1.7, and 1.0 kilobases. Southern analysis indicated that the DNA sequence encoding the HSV-specific uracil-DNA glycosylase was located between 0.065 and 0.08 map units on the prototypic arrangement of the HSV genome.
Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) (EC 3.6.1.23) derived from HeLa S3 cells has been purified to near homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme has a specific activity of about 16,000 nmol of dUMP hydrolyzed per min/mg of protein. The dUTPase enzyme derived from HeLa S3 cells appears to be composed to two equal molecular mass subunits, each being about 22,500 daltons. Association of these subunits to produce a 45,000-dalton protein is promoted by MgCl2. In the presence of EDTA enzyme activity is abolished and the enzyme dissociates into its monomeric form. MgCl2 will completely reverse the inhibition caused by EDTA and promote subunit association. MnCl2 will also promote association of the dUTPase subunits. However, MnCl2 will not completely reverse inhibition by EDTA. In addition, purified dUTPase, extensively dialyzed to remove contaminating ions, is activated almost 2-fold by the addition of 5 mM MgCl2. In contrast, addition of 5 mM MnCl2 to the dialyzed enzyme preparation will cause more than a 50% decrease in enzyme activity. This data indicates that Mg2+ is the natural prosthetic group for this enzyme. The Km value of dUTP for the purified enzyme is 3 X 10(-6) M in the presence of MgCl2. The turnover number for this enzyme has been calculated to be 550 molecules of dUTP hydrolyzed per min under standard assay conditions. Infection of HeLa S3 cells with herpes simplex type 1 virus induces a distinct species of dUTPase. This new species of dUTPase has an isoelectric point of 8.0, compared to an isoelectric point in the range of 5.7 to 6.5 for the HeLa S3 dUTPase. Molecular weight determinations of this new species of dUTPase indicate that the native enzyme is monomeric with a molecular weight of about 35,000. The virally induced dUTPase is inhibited by EDTA and this inhibition is reversed by MgCl2. Unlike the HeLa S3 dUTPase, the virally induced enzyme does not appear to be composed of subunits.
Monoclonal antibodies specific for herpes simplex virus type 1 (HSV-1) glycoproteins were used to demonstrate that HSV undergoes mutagen-induced and spontaneous antigenic variation. Hybridomas were produced by polyethylene glycol-mediated fusion of P3-X63-Ag8.653 myeloma cells with spleen cells from BALB/c mice infected with HSV-1 (strain KOS). Hybrid clones were screened for production of HSV-specific neutralizing antibody. The glycoprotein specificities of the antibodies were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates of radiolabeled infected-cell extracts. Seven hybridomas producing antibodies specific for gC, one for gB, and one for gD were characterized. All antibodies neutralized HSV-1 but not HSV-2. Two antibodies, one specific for gB and one specific for gC, were used to select viral variants resistant to neutralization by monoclonal antibody plus complement. Selections were made from untreated and bromodeoxyuridine- and nitrosoguanidine-mutagenized stocks of a plaque-purified isolate of strain KOS. After neutralization with monoclonal antibody plus complement, surviving virus was plaque purified by plating at limiting dilution and tested for resistance to neutralization with the selecting antibody. The frequency of neutralization-resistant antigenic variants selected with monoclonal antibody ranged from 4 X 10(-4) in nonmutagenized stocks to 1 X 10(-2) in mutagenized stocks. Four gC and four gB antigenic variants were isolated. Two variants resistant to neutralization by gC-specific antibodies failed to express gC, accounting for their resistant phenotype. The two other gC antigenic variants and the four gB variants expressed antigenically altered glycoproteins and were designated monoclonal-antibody-resistant, mar, mutants. The two mar C mutants were tested for resistance to neutralization with a panel of seven gC-specific monoclonal antibodies. The resulting patterns of resistance provided evidence for at least two antigenic sites on glycoprotein gC.
The enzyme uracil DNA-glycosylase has been purified from blast cells of patients with acute myelocytic leukemia. A 1000-fold purification has been achieved and the enzyme appears highly enriched for the uracil glycosylase activity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular weight of the purified enzyme is 30,000. Uracil DNA-glycosylase exhibits activity in the absence of any added metal and the addition of MgCl2, MnCl2, CaCl2, NaCl, or KCl causes inhibition. EDTA as well as EGTA can inhibit enzyme activity. An interesting finding is the biphasic effect of spermine. At a concentration of 25 microM, spermine will cause a 2.5-fold activation of enzyme activity, whereas at concentrations of 100 microM and higher, spermine will inhibit enzyme activity. An Arrhenius plot of glycosylase activity in the presence of 25 microM spermine shows a biphasic curve with the transition temperature being 36 degrees C. Initial velocity studies in the presence of varying concentrations of spermine indicate a change in both the apparent Km and Vmax of the enzyme. Various uracil analogs were tested to establish a structure-activity relationship for this enzyme. It appears from this data that uracil DNA-glycosylase is very specific for uracil moieties. Uracil, acting as a product inhibitor, gives a Ki value of 220 microM.
The cell cycle stimulation of individual species of the uracil DNA glycosylase was examined in WI-38 normal diploid fibroblasts. The nuclear uracil DNA glycosylase was induced as WI-38 cells traversed the cell cycle. In contrast, the specific activity of the mitochondrial glycosylase remained constant during cell proliferation. The two enzyme activities can be further distinguished by their elution patterns on DNA-cellulose, by differential cation sensitivity, and by kinetic differences. The singular stimulation of the nuclear glycosylase in the cell cycle is a further suggestion that normal human cells actively regulate excision repair pathways.
This book presents immuno-chemical techniques for isolating and analysing a wide variety of biological products. CONTENTS: Introduction, Polyclonal Antiserum Production;Polyclonal Antiserum Processing;Uses of Polyclonal Antisera. Production and Use of Monoclonal Antibodies;Synthetic Petpides: A New Development in Protein Immunochemistry;and Technical Supplement.
An improved method is described for the renaturation of microgram amounts of proteins from sodium dodecyl sulfate-polyacrylamide gels. The protein band is visualized in the gel by KCl staining, the band cut out and crushed, and the protein eluted by diffusion in a buffer containing 0.1% sodium dodecyl sulfate. Protein is concentrated and sodium dodecyl sulfate is removed by acetone precipitation of the sample. Renaturation of the protein occurs after the precipitate is dissolved in 6 m guanidine hydrochloride and then diluted. The activity of the sigma subunit of Escherichia coli RNA polymerase can be recovered with 98–100% efficiency after electrophoresis in an SDS-gel and renaturation by this technique. To assess whether the method is generally applicable we tested some or all of the steps involved in the procedure using E. coli transcription termination factor rho, β-galactosidase, alkaline phosphatase, wheat α-amylase, and DNA topoisomerase. We show how the method can be used to determine the approximate molecular weight of the DNA topoisomerase polypeptide by sectioning a gel on which a partially pure sample has been fractionated by electrophoresis.
This chapter discusses that since DNA is the carrier of genetic information and spontaneous mutations occur only at low frequency, cellular DNA has often been regarded as an essentially stable entity. Recent developments have necessitated a revision of this view. With the discovery of insertion elements, it became clear that certain segments of DNA can move between many different chromosomal sites. Further, the susceptibility of DNA to heat-induced degradation at moderate temperatures and neutral pH leads to hydrolytic decay at a much faster rate than that expected from spontaneous mutation frequencies. The latter, somewhat paradoxical, observation can be rationalized by postulating the existence of efficient repair mechanisms to maintain the integrity of DNA. The chapter also discusses that several enzymes that act specifically on hydrolytically-damaged nucleotide residues in DNA have recently been discovered, purified, and characterized, and they are the main subject of the present review. Some of these enzymes, the DNA glycosylases, belong to a previously unrecognized class of enzymes that cleave base–sugar bonds in DNA. In addition to their role in surveying and removing DNA damage that would otherwise lead to unacceptable spontaneous mutation frequencies, the same enzymes may also play an important role in the repair of cellular lesions introduced by ionizing radiation or by exposure to chemical mutagens such as alkylating agents, nitrous acid, or bisulfite.
Tumor suppressor genes are wild-type alleles of genes that play regulatory roles in cell proliferation, differentiation, and
other cellular and systemic processes. It is their loss or inactivation that is oncogenic. The first evidence of tumor suppressor
genes appeared in the early 1970s, but only within the past few years has a wealth of new information illuminated the central
importance of these genes. Two or more different suppressor genes may be inactivated in the same tumors, and the same suppressors
may be inactive in different tumor types (for example, lung, breast, and colon). The suppressor genes already identified are
involved in cell cycle control, signal transduction, angiogenesis, and development, indicating that they contribute to a broad
array of normal and tumor-related functions. It is proposed that tumor suppressor genes provide a vast untapped resource for
anticancer therapy.
Uracil-DNA glycosylase has been purified approximately 130,000-fold from extracts of human placenta. Although all of the uracil-DNA glycosylase activity coeluted through six chromatographic steps, at least four distinct peaks of activity were resolved in the final purification on a Mono S column. Each of the peaks containing uracil-DNA glycosylase activity contained two peptides of Mr = 29,000 and Mr = 26,500, respectively, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Experimental evidence indicated that the Mr = 29,000 peptide was the uracil-DNA glycosylase enzyme. The amino-terminal sequence of each peptide was determined after blotting of the peptides from the gel onto Polybrene GF/C paper. The sequences were not related to each other, and neither was any significant homology to other proteins found. Uracil-DNA glycosylase had a molecular turnover number of approximately 600/min and apparent Km value of 2 microM. The enzyme is a basic protein and was stimulated about 10-fold by 60-70 mM NaCl whereas higher concentrations were inhibitory.
It has been reported that Drosophila lacks a uracil-DNA glycosylase but that a direct incising activity on uracil-containing DNA appeared developmentally only in third instar larvae. In contrast we have found by two independent assays, that uracil-DNA glycosylase exists in both Drosophila eggs as well as in third instar larvae. The first assay shows the liberation of [3H] uracil from a d(AT)n polymer randomly substituted with [3H]uracil by its synthesis in the presence of [3H] dUTP. The second fluorometric assay for uracil-DNA glycosylase depends on the unique topological properties of circular DNAs and has the advantage of detecting apyrimidinic/apurinic (AP) endonuclease activity as well. To test one other insect, locust eggs were also assayed for uracil-DNA glycosylase. The amount of uracil-DNA glycosylase correlated well with the amount of DNA in actively replicating cells.
Uracil-DNA glycosylase from rat liver mitochondria, an inner membrane protein, has been purified approximately 575,000-fold to apparent homogeneity. During purification two distinct activity peaks, designated form I and form II, were resolved by phosphocellulose chromatography. Form I constituted approximately 85% while form II was approximately 15% of the total activity; no interconversion between the forms was observed. The major form was purified as a basic protein with an isoelectric point of 10.3. This enzyme consists of a single polypeptide with an apparent Mr of 24,000 as determined by recovering glycosylase activity from a sodium dodecyl sulfate-polyacrylamide gel. A native Mr of 29,000 was determined by glycerol gradient sedimentation. The purified enzyme had no detectable exonuclease, apurinic/apyrimidinic endonuclease, DNA polymerase, or hydroxymethyluracil-DNA glycosylase activity. A 2-fold preference for single-stranded uracil-DNA over a duplex substrate was observed. The apparent Km for uracil residues in DNA was 1.1 microM, and the turnover number is about 1000 uracil residues released per minute. Both free uracil and apyrimidinic sites inhibited glycosylase activity with Ki values of approximately 600 microM and 1.2 microM, respectively. Other uracil analogues including 5-(hydroxymethyl)uracil, 5-fluorouracil, 5-aminouracil, 6-azauracil, and 2-thiouracil or analogues of apyrimidinic sites such as deoxyribose and deoxyribose 5'-phosphate did not inhibit activity. Both form I and form II had virtually identical kinetic properties, and the catalytic fingerprints (specificity for uracil residues located in a defined nucleotide sequence) obtained on a 152-nucleotide restriction fragment of M13mp2 uracil-DNA were almost identical. These properties differentiated the mitochondrial enzyme from that of the uracil-DNA glycosylase purified from nuclei of the same source.
Human placental uracil DNA glycosylase was purified 3700-fold to apparent homogeneity as defined by SDS gel analysis. Its immunological characteristics were examined using three monoclonal antibodies prepared against partially purified human placental uracil DNA glycosylase. Immunoblot analysis demonstrated that, even in crude isolates, only one glycosylase species of molecular weight 37,000 could be detected. Each of the three monoclonal antibodies quantitatively recognized the highly purified enzyme by ELISA. The glycosylase is a single polypeptide with a molecular weight of 37,000 as defined by both Sephadex gel filtration and by SDS-polyacrylamide gel electrophoresis analysis. The enzyme is heat-stable, with a t 1/2 of greater than 30 min at 42 degrees C or at 45 degrees C. Surprisingly, inhibitor analysis demonstrated that the glycosylase was inhibited by preincubation with either 5-fluorouracil or 5-bromouracil. However, no significant inhibition was observed when either compound was added directly to the enzyme assay.
Rat liver uracil-DNA glycosylase has been purified from nuclear extracts over 3000-fold to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is a monomeric protein with a polypeptide molecular weight of approximately 35 000. It has a native molecular weight of 33 000 as determined by gel filtration chromatography and a sedimentation coefficient of 2.6 S in glycerol gradients. The nuclear enzyme has an alkaline pH optimum and a pI value of 9.3. Nuclear uracil-DNA glycosylase catalyzes the release of free uracil from both single-stranded and double-stranded DNA with the former being the preferred substrate. The enzyme is unable to recognize dUTP, dUMP, or poly(dA-dT) containing a 3'-terminal uracil residue as a substrate. However, internalization of terminal uracil residues by limited chain elongation produced a substrate for the glycosylase. Another species of uracil-DNA glycosylase has been partially purified from mitochondria. This activity differs from the nuclear enzyme in that it has (i) distinctive chromatographic properties, (ii) a lower native molecular weight of 20 000 as determined by molecular sieving, (iii) a distinct NaCl inhibition profile, and (iv) a longer half-life during thermal denaturation.
By analyzing the effects of single base substitutions around the ATG initiator codon in a cloned preproinsulin gene, I have identified ACCATGG as the optimal sequence for initiation by eukaryotic ribosomes. Mutations within that sequence modulate the yield of proinsulin over a 20-fold range. A purine in position -3 (i.e., 3 nucleotides upstream from the ATG codon) has a dominant effect; when a pyrimidine replaces the purine in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Single base substitutions around an upstream, out-of-frame ATG codon affect the efficiency with which it acts as a barrier to initiating at the downstream start site for preproinsulin. The optimal sequence for initiation defined by mutagenesis is identical to the consensus sequence that emerged previously from surveys of translational start sites in eukaryotic mRNAs. The mechanism by which nucleotides flanking the ATG codon might exert their effect is discussed.
In this paper we describe the synthesis and cloning of a short segment of DNA complementary to the region immediately adjacent
to the EcoRI insertion site in the single-stranded bacteriophage vector M13mp2. This segment is useful as a “universal” primer for DNA
sequencing by the dideoxynucleotide chain termination method; the template can be any DNA species cloned in M13mp2 or its
derivatives. The primer has been cloned into the tetracycline resistance gene of plasmid pBR322 as one strand of a 26 bp EcoRI/BamHI fragment. This fragment may be readily prepared from an EcoRI + BamHI restriction digest of the parent plasmid (designated pSPi4) by a simple size fractionation.
A method for producing random subclones using sonication to fragment the DNA is presented. The sonication is combined with enzymatic repair of the fragment ends and a rigorous size fractionation step to prepare subclones of relatively homogeneous and specific size. Under some conditions sonication is shown to shear A + T-rich sequences preferentially, although under most conditions it will create a random subclone library. The use of these subclone libraries for an improved "shotgun" DNA sequencing strategy is tested on a 17.2-kb (kilobase) fragment of Epstein-Barr virus.
The University of Wisconsin Genetics Computer Group (UWGCG) has been organized to develop computational tools for the analysis
and publication of biological sequence data. A group of programs that will interact with each research-article has been developed
for the Digital Equipment Corporation VAX computer using the VMS operating system. The programs available and the conditions
for transfer are described.
An activity which releases free uracil from bacteriophage PBS1 DNA has been purified over 10,000 fold from extracts of Saccharornyces cerevisiae. The enzyme is active on both native and denatured PBSl UNA and is active in the absence of divalent cation, and in the presence
of 1 mM EDTA. The enzyme has a native molecular weight of 27,800 as estimated by glycerol gradient centrifugation and gelfiltration.
Enzyme activity has been recovered after denaturation in SDS and electrophoresis in an SDS polyacrylamide gel. This analysis
suggests that the enzyme consists of a single polypeptide chain of about 27,000 daltons. Normal levels of uracil-DNA glycosylase
activity were found in partially purified extracts of the nitrous-acid sensitive rad18-2 mutant of yeast.