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Purification and determination of the NH2-terminal amino acid sequence of uracil-DNA glycosylase from human placenta
Abstract
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.
... Recently, uracil-DNA glycosylase from human placenta was purified and partially sequenced (23), the corresponding cDNA (UNG15 and UNG40) was cloned (24), and the gene assigned to chromosome 12 (25). A strong sequence similarity was observed to UDG from E. coli (26), Streptococcus pneumoniae (27), animal viruses (28-31) and yeast (32), ranging from 41 to 56% identical residues. ...
... 1.5 ,tl was used in each translation experiment in a total of 10 ,tl, and the potassium acetate concentration was reduced to 27 mM to compensate for the potassium ions from the hybrid arrest mixture. Translation was of uracil-DNA glycosylase identified in uracil-DNA glycosylase prepared from human placenta was Ala-Arg-Asn and the protein had an apparent molecular mass of 29 kD as determined by SDSpolyacrylamide electrophoresis (23). Ala-Arg-Asn was found in the amino acid sequence predicted from UNG15 at a position 78 amino acids downstream of the NH2-terminal. ...
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.
... Until now, little information has existed on the enzymatic properties of full-length hUNG2. A likely reason for this is the susceptibility of the enzyme to N-terminal proteolytic degradation during purification, mainly resulting in the core catalytic domain (25). To gain further insight in the functional properties of hUNG2 and hSMUG1 and their relative contribution to nuclear base excision repair, both proteins were purified after overexpression in E. coli. ...
... These results indicate that the hUNG2 N-terminal regulatory region constitutes a distinct domain sensitive to proteolysis. This would also explain why previous attempts to purify UNG from mammalian cells in the absence of appropriate protease inhibitors have yielded essentially the catalytic domain (25). This is also corroborated by the abnormal chromatographic behavior of hUNG2. ...
hUNG2 and hSMUG1 are the only known glycosylases that may remove uracil from both double- and single-stranded DNA in nuclear chromatin, but their relative contribution to base excision repair remains elusive. The present study demonstrates that both enzymes are strongly stimulated by physiological concentrations of Mg2+, at which the activity of hUNG2 is 2-3 orders of magnitude higher than of hSMUG1. Moreover, Mg2+ increases the preference of hUNG2 toward uracil in ssDNA nearly 40-fold. APE1 has a strong stimulatory effect on hSMUG1 against dsU, apparently because of enhanced dissociation of hSMUG1 from AP sites in dsDNA. hSMUG1 also has a broader substrate specificity than hUNG2, including 5-hydroxymethyluracil and 3,N(4)-ethenocytosine. hUNG2 is excluded from, whereas hSMUG1 accumulates in, nucleoli in living cells. In contrast, only hUNG2 accumulates in replication foci in the S-phase. hUNG2 in nuclear extracts initiates base excision repair of plasmids containing either U:A and U:G in vitro. Moreover, an additional but delayed repair of the U:G plasmid is observed that is not inhibited by neutralizing antibodies against hUNG2 or hSMUG1. We propose a model in which hUNG2 is responsible for both prereplicative removal of deaminated cytosine and postreplicative removal of misincorporated uracil at the replication fork. We also provide evidence that hUNG2 is the major enzyme for removal of deaminated cytosine outside of replication foci, with hSMUG1 acting as a broad specificity backup.
... With the help of Dr. Guy Bauw, we succeeded in obtaining N-terminal sequences for both peptides in the preparation. One of these, of 29 kDa, was our candidate protein for uracil-DNA glycosylase [28]. We had delayed the publication for a couple of years until we were ready to start cloning of cDNA. ...
... Assay for testing substrate speci®cities of engineered enzymes Two hundred and ®fty nanograms of enzyme (10 pmol, UDG, CDG or TDG) were incubated with 17±39 pmol of 3 H-labelled (A, C, G, T or U) substrate DNA, at 37°C for 3 h under standard conditions (10 mM Tris±HCl pH 7.5, 1 mM EDTA, 10 mM NaCl, 50 mg/ml bovine serum albumin) (Kavli et al., 1996), and excision activity was measured as previously described (Wittwer et al., 1989). ...
We introduced multiple abasic sites (AP sites) in the chromosome of repair-deficient mutants of Escherichia coli, in vivo, by expressing engineered variants of uracil-DNA glycosylase that remove either thymine or cytosine. After introduction of AP sites, deficiencies in base excision repair (BER) or recombination were associated with strongly enhanced cytotoxicity and elevated mutation frequencies, selected as base substitutions giving rifampicin resistance. In these strains, increased fractions of transversions and untargeted mutations were observed. In a recA mutant, deficient in both recombination and translesion DNA synthesis (TLS), multiple AP sites resulted in rapid cell death. Preferential incorporation of dAMP opposite a chromosomal AP site ('A rule') required UmuC. Furthermore, we observed an 'A rule-like' pattern of spontaneous mutations that was also UmuC dependent. The mutation patterns indicate that UmuC is involved in untargeted mutations as well. In a UmuC-deficient background, a preference for dGMP was observed. Spontaneous mutation spectra were generally strongly dependent upon the repair background. In conclusion, BER, recombination and TLS all contribute to the handling of chromosomal AP sites in E.coli in vivo.
... The molecular mass of cUNG was determined to 25 kDa. This is approximately the same molecular mass as the UNG purified from human placenta (29 kDa, Wittwer et al., 1989) and the rhUNG (UNGD84, 27 kDa), which lacked 77 and 84 of the first N-terminal amino acids, respectively, as predicted from the mitochondrial ORF (Slupphaug et al., 1995). This suggests that the N-terminal signal sequence in the purified cUNG is processed or artificially cleaved during purification or that Atlantic cUNG lacks an N-terminal signal sequence. ...
Uracil-DNA glycosylase (UDG; UNG) has been purified 17000-fold from Atlantic cod liver (Gadus morhua). The enzyme has an apparent molecular mass of 25 kDa, as determined by gel filtration, and an isoelectric point above 9.0. Atlantic cUNG is inhibited by the specific UNG inhibitor (Ugi) from the Bacillus subtilis bacteriophage (PBS2), and has a 2-fold higher activity for single-stranded DNA than for double-stranded DNA. cUNG has an optimum activity between pH 7.0-9.0 and 25-50 mM NaCl, and a temperature optimum of 41 degrees C. Cod UNG was compared with the recombinant human UNG (rhUNG), and was found to have slightly higher relative activity at low temperatures compared with their respective optimum temperatures. Cod UNG is also more pH- and temperature labile than rhUNG. At pH 10.0, the recombinant human UNG had 66% residual activity compared with only 0.4% for the Atlantic cUNG. At 50 degrees C, cUNG had a half-life of 0.5 min compared with 8 min for the rhUNG. These activity and stability experiments reveal cold-adapted features in cUNG.
... High-turnover activity assays using [3H]-labeled nick translated calf thymus DNA substrates (43) were performed essentially as described previously (12). In brief, activity was measured in 20 μl assay mixture containing (final) 20 mM Tris-HCl, pH 7.5, 60 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.5 mg/ml BSA, 36 pmol [3H]dUMP-labeled calf thymus DNA substrate and 2 μg extract (measured as total protein) at 30°C for 10 min (or 4 μg protein, 37°C for 30 min in high-sensitivity assays). ...
The generation of high-affinity antibodies requires somatic hypermutation (SHM) and class switch recombination (CSR) at the immunoglobulin (Ig) locus. Both processes are triggered by activation-induced cytidine deaminase (AID) and require UNG-encoded uracil-DNA glycosylase. AID has been suggested to function as an mRNA editing deaminase or as a single-strand DNA deaminase. In the latter model, SHM may result from replicative incorporation of dAMP opposite U or from error-prone repair of U, whereas CSR may be triggered by strand breaks at abasic sites. Here, we demonstrate that extracts of UNG-proficient human B cell lines efficiently remove U from single-stranded DNA. In B cell lines from hyper-IgM patients carrying UNG mutations, the single-strand-specific uracil-DNA glycosylase, SMUG1, cannot complement this function. Moreover, the UNG mutations lead to increased accumulation of genomic uracil. One mutation results in an F251S substitution in the UNG catalytic domain. Although this UNG form was fully active and stable when expressed in Escherichia coli, it was mistargeted to mitochondria and degraded in mammalian cells. Our results may explain why SMUG1 cannot compensate the UNG2 deficiency in human B cells, and are fully consistent with the DNA deamination model that requires active nuclear UNG2. Based on our findings and recent information in the literature, we present an integrated model for the initiating steps in CSR.
Background:
Heat-labile uracil-DNA glycosylase (HL-UDG) is commonly employed to eliminate carry-over contamination in DNA amplifications. However, the prevailing HL-UDG is markedly inactivated at 50°C, rendering it unsuitable for specific one-step RT-qPCR protocols utilizing reverse transcriptase at an optimal temperature of 42°C.
Objective:
This study aimed to explore novel HL-UDG with lower inactivation temperature and for recombinant expression.
Methods:
The gene encoding an HL-UDG was cloned from the cold-water fish rainbow trout (Oncorhynchus mykiss) and expressed in Escherichia coli with high yield. The thermostability of the enzyme and other enzymatic characteristics were thoroughly examined. The novel HL-UDG was then applied for controlling carry-over contamination in one-step RT-qPCR.
Results:
This recombinantly expressed truncated HL-UDG of rainbow trout (OmUDG) exhibited high amino acids similarity (84.1% identity) to recombinant Atlantic cod UDG (rcUDG) and was easily denatured at 40°C. The optimal pH of OmUDG was 8.0, and the optimal concentrations of both Na+ and K+ were 10 mM. Since its inactivation temperature was lower than that of rcUDG, the OmUDG could be used to eliminate carry-over contamination in one-step RT-qPCR with moderate reverse transcription temperature.
Conclusion:
We successfully identified and recombinantly expressed a novel HL-UDG with an inactivation temperature of 40°C. It is suitable for eliminating carry-over contamination in one-step RT-qPCR.
Transcripts of many enzymes involved in base excision repair (BER) undergo extensive alternative splicing, but functions of the corresponding alternative splice variants remain largely unexplored. In this review, we cover the studies describing the common alternatively spliced isoforms and disease-associated variants of DNA glycosylases, AP-endonuclease 1, and DNA polymerase beta. We also discuss the roles of alternative splicing in the regulation of their expression, catalytic activities, and intracellular transport.
Uracil in DNA results from either misincorporation of dUMP residues during replication, or from deamination of cytosine residues. The latter process results in premutagenic U:G mispairs that, unless uracil is removed, will cause GC→AT transitions in the subsequent round of replication. The 13.8 kb gene for human uracil-DNA glycosylase, UNG, is highly conserved and comprises 7 exons. It encodes more than 98% of the total uracil-DNA glycosylase activity in the cell. The crystal structure of the catalytic domain of UNG in complex with target DNA has demonstrated that all essential contacts are with the uracil-containing strand. The structure also reveals the mechanism of enzyme-assisted flipping of the uracil-containing nucleotide into the deep catalytic pocket that specifically binds uracil. Nuclear (UNG2) and mitochondrial (UNG1) forms of the enzyme result from the use of two promoters, PA and PB, and alternative splicing. mRNA for UNG1 encodes 304 amino acids, the first 35 of which are unique to this form. mRNA for UNG2 encodes 313 amino acids, the first 44 of which are unique to UNG2. The unique N-terminal sequences in UNG1 and UNG2 are required for mitochondrial and nuclear sorting, respectively, but not for catalytic activity. The 269 amino acid residues common to the two forms include the compact catalytic domain of approximately 220 C-terminal residues and an N-terminal part that binds replication protein A (RPA), indicating a possible role for RPA in base excision repair.
This article will mainly review the work of our laboratories on the repair of DNA damage in human cells and tissues and will include repair of damage caused by ultraviolet light, uracil in DNA and alkylations due to Nnitroso compounds. References to related work will be restricted to those pertinent to the discussion and interpretation of our own work.
A study was made of the dependence of the affinity of oligonucleotides d(pC)n, d(pT)n, and d(pA)n, and some duplexes to apurinic/apyrimidinic endonuclease on their length. Simple algorithms were developed for estimating the affinity of single-stranded oligonucleotides and their duplexes to this enzyme. Nucleoside monophosphates (dNMPs) were shown to be the minimal ligands for AP endonuclease (Ki = 420-500 μM). The enzyme interacts with 10 units of a DNA ligand regardless of its length. Raising the oligonucleotide length by one unit (at n 10) increases its affinity (factor f) 1.5-1.6-fold. The affinity increases according to the progression Ki[d(pN)n] = Ki[dNMP]*f1-n. The affinity of d(pA)n d(pT)n changes in a similar way, f = 1.95. Introduction of an apurinic site into single-stranded oligonucleotides and complementary duplexes increases their affinity only 3-10-fold, suggesting that recognition of apurinic sites by the enzyme does not play a key role in its interaction with modified DNA. Recognition of the substrate takes place basically through weak additive interactions of the enzyme with nine intact pairs.
We have investigated the specificities of G.T mismatch binding proteins and of G.T mismatch cleavage in extracts of mammalian cells. G.T mismatch-specific protein:DNA complex formation by cell extracts was independent of the local sequence context of the mismatch. Cell extracts performed similar levels of protein binding to DNA substrates in which a single G.T mispair was preceded by T, G, A, C, or 5-meC. In contrast, incision by extracts of the T-containing strand of a G.T mismatch exhibited a strong sequence specificity and efficient strand cleavage was only observed when the mismatched G was in a CpG sequence. Thus, oligonucleotides containing either CpG/GpT or 5meCpG/GpT were efficiently incised, but not those containing GpG/CpT, ApG/TpT, or TpG/ApT sequences. Cell lines made resistant to the alkylating agent N-methyl-N-nitrosourea have previously been found to be defective in a G.T mismatch binding reaction. The defect in binding by extracts prepared from these cells extended to G.T mismatches in several sequence contexts. The variant extracts nevertheless incised G.T mismatches normally suggesting that this particular binding activity is not required for incision. The data indicate that incision by this activity is targeted to the CpG sequences in which G.T mismatches are formed by the mutagenic deamination of DNA 5-methylcytosine. In this regard the repair pathway resembles the very short patch (vsp) repair pathway in Escherichia coli.
A structural homology between the endogenous differentiation factor of the HL-60 cell line of promyelocyte leukemia (HLDF)
and several DNA/RNA-binding and DNA/RNA-hydrolyzing proteins was revealed, and expression of thehldf gene in prokaryotic systems was studied. On the basis of these experiments, the amino acid sequence of an 8-membered fragment
of HLDF with potential nuclease activity was identified. The synthetic octapeptide RRWHRLKE was shown to be capable of the
cleavage of RNA, linear DNA from phage λ, and all forms of plasmid DNA. We established that treatment of the HL-60 cell culture
with this peptide (10−6 M) results in an increase in the number of apoptotic cells and suggested that HLDF is involved in processes of apoptosis.
Studies on the enzymology of apurinic/apyrimidinic (AP) endonucleases from procaryotic and eucaryotic organisms are reviewed. Emphasis will be placed on the enzymes from Escherichia coli from which a considerable portion of our knowledge has been derived. Recent studies on similar enzymes from eucaryotes will be discussed as well. In addition, we will discuss the chemical and physical properties of AP sites and review studies on peptides and acridine derivatives which incise DNA at AP sites.
Bloom's syndrome uracil DNA glycosylase was highly purified from two non-transformed cell strains derived from individuals from different ethnic groups. Their properties were then compared to two different highly purified normal human uracil DNA glycosylases. A molecular mass of 37 kDa was observed for each of the four human enzymes as defined by gel-filtration column chromatography and by SDS-PAGE. Each of the 37 kDa proteins was identified as a uracil DNA glycosylase by electroelution from the SDS polyacrylamide gel, determination of glycosylase activity by in vitro biochemical assay and identification of the reaction product as free uracil by co-chromatography with authentic uracil. Bloom's syndrome enzymes differed substantially in their isoelectric point and were thermolabile as compared to the normal human enzymes. Bloom's syndrome enzymes displayed a different Km, Vmax and were strikingly insensitive to 5-fluorouracil and 5-bromouracil, pyrimidine analogues which drastically decreased the activity of the normal human enzymes. In particular, each Bloom's syndrome enzyme required 10-100-fold higher concentrations of each analogue to achieve comparable inhibition of enzyme activity. Potential mechanisms are considered through which an altered uracil DNA glycosylase characterizing this cancer-prone human genetic disorder may arise.
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.
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.
An apparently homogeneous enzyme preparation, containing an apurinic/apyrimidinic (AP) endonuclease from human placenta, was
by DNA sequencing analysis found to act as a class I AP-endonuclease, i.e. produce a 3'-deoxyribose and 5'-phosphomonoester
nucleotide termini.
Topics covered in this book included: Eukaryote model systems for DNA repair study; Sensitive detection of DNA lesions and their repair; and Defined DNA sequence probes for analysis of mutagenesis and repair.
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.
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 subcellular localization of the human DNA-repair enzyme uracil-DNA glycosylase from the UNG gene has been studied using flow cytometry and laser scanning confocal microscopy of freely cycling HeLa S3 cells. A two-parameter flow cytometric analysis using propidium iodide and UNG-specific antibodies demonstrated that total cellular UNG increased during the G1-phase and was approximately doubled in early S-phase compared to early G1. The UNG level was stable during the S-phase and increased further during G2, reaching a 2.8-fold level compared to early G1. This factor included differences in cell size and staining variabilities. These findings were confirmed using two-parameter confocal analysis of UNG/DNA and UNG/mitochondria at different stages of the cell cycle. Although the major fraction of UNG was associated with nuclei, we also observed distinctive staining associated with mitochondria and a more diffuse staining probably reflecting UNG in the cytosol. Furthermore, very little UNG staining was observed in nucleoli. The UNG level in different cell compartments varied at different stages of the cell cycle, and this variation was most pronounced in the nuclei. These results demonstrate that the gene product from the UNG gene is located within three subcellular compartments and that the distribution between these compartments varies during the cell cycle.
In order to study the mechanism of action of uracil-DNA glycosylase (UDG) from human placenta, single-stranded (ss) and double-stranded (ds) oligodeoxyribonucleotides (oligos), containing deoxyuridine (dU) and a wide variety of their analogs were used. It was shown that UDG has a twofold preference for ss oligos over ds oligos and a twofold preference for intermolecular duplexes over similar hairpin-like duplexes. The replacement of dU with 1-(beta-D-2'-deoxy-threo-pentofuranosil)uracil (xU) or 1-(beta-D-3'-deoxy-threo-pentofuranosil)uracil (tU), which results in a change in sugar hydroxyl configuration, has no influence on UDG binding to such substrates, but inhibits uracil removal. A oligo containing 2'-deoxy-2'-fluorouridine (flU), with a 3'-endo conformation of modified sugar is recognized by UDG 100-200-fold less efficiently than the natural ones. F or Br atoms or a methyl group were introduced at position 5 of a dU residue in an oligo. It was shown that the nature of a substituent at this position is essential for UDG function.
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.
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.
The gene for human uracil-DNA glycosylase (UNG) contains 4 exons and has an approximate size of 13 kb. The promoter is very GC rich and lacks a TATA box. Nested deletions of the promoter demonstrated that two SP1 elements and a putative c-MYC element proximal to the transcription initiation region were sufficient to support some 27% of the promoter activity, while a clone that in addition contained the elements E2F/SP1/CCAAT increased expression to almost 90% of the full-length construct. A region upstream of these elements appears to exert a negative control function.
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.
The bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) inactivates Escherichia coli uracil-DNA glycosylase (Ung) by forming an Ung.Ugi protein complex with 1:1 stoichiometry. Stability of the Ung.Ugi complex was demonstrated by the inability of free Ugi to exchange with Ugi bound in preformed complex. Ung was reacted with fluorescein 5-isothiocyanate to produce fluorescent-Ung (F-Ung), which retained full uracil-DNA glycosylase activity and susceptibility to Ugi inactivation. Addition of Ugi to F-Ung under steady-state conditions resulted in saturable (15%) fluorescence quenching at a F-Ung.Ugi ratio of 1:1.4. Dissociation constants determined for the F-Ung interaction with M13 DNA, uracil-containing DNA, and poly(U) equaled 600, 220, and 190 microM, respectively. While F-Ung associated with nucleic acid polymers was able to bind Ugi efficiently, F-Ung bound in the F-Ung.Ugi complex could no longer effectively bind nucleic acid. Stopped-flow kinetic analysis suggested the F-Ung/Ugi association was described by a two-step mechanism. The first step entailed a rapid pre-equilibrium distinguished by the dissociation constant Kd = 1.3 microM. The second step led irreversibly to the formation of the final complex and was characterized by the rate constant k = 195 s-1. We infer Ugi inactivates Ung through the formation of an exceptionally stable protein-protein complex.
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.
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.
Two clones containing nonfunctional pseudogenes for the human uracil-DNA glycosylase gene have been isolated. The sequences of the two clones that are homologous to the UNG cDNA span 670 and 580 bp, respectively. In the longest of these, a full length Sx type Alu sequence interrupts the homologous sequence. Chromosomal mapping locates the clones to chromosomes 16 and 14. Comparison of the pseudogene sequences to the cDNA sequence indicates that the pseudogenes diverged from the functional gene approximately 31 and 22 million years ago, which is before the point in evolution when great apes and hominides separated.
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.
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).
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.
A procedure for isolating electrophoretically homogeneous apurinic/apyrimidinic (AP) endonuclease from human placenta was elaborated. Enzyme preparations contained no impurities that cleave nucleic acids. The enzyme cleaves single-and double-stranded oligonucleotides lacking one or two bases. For a number of oligonucleotides containing one or two AP sites at various positions in one or both strands, KM and Vmax were estimated and their dependence on the position of AP site in the duplex was elucidated. Cleavage of single-stranded oligonucleotides also slightly depended on their nucleotide sequence and the position of AP site. Enzyme-induced active DNA conformation was assumed to be the major factor in the increased rate and the specificity of cleavage.
The preform of human mitochondrial uracil-DNA glycosylase (UNG1) contains 35 N-terminal residues required for mitochondrial
targeting. We have examined processing of human UNG1 expressed in insect cells and processing in vitro by human mitochondrial extracts. In insect cells we detected a major processed form lacking 29 of the 35 unique N-terminal
residues (UNG1Δ29, 31 kDa) and two minor forms lacking the 75 and 77 N-terminal residues, respectively (UNG1Δ75 and UNG1Δ77,
26 kDa). Purified UNG1Δ29 was effectively cleaved in vitro to a fully active 26 kDa form by human mitochondrial extracts. Furthermore, endogenous forms of 31 and 26 kDa were also observed
in HeLa mitochondrial extracts. The sequences at the cleavage sites, as identified by peptide sequencing, were compatible
with the known specificity of mitochondrial processing peptidase (MPP). However, in vitro cleavage of UNG1Δ29 by mitochondrial extracts did not require divalent cations and was stimulated by EDTA, indicating the
involvement of a processing peptidase distinct from MPP at the second site. Interestingly, while UNG1Δ29 generally has the
typical properties reported for other uracil-DNA glycosylases, it is not inhibited by apurinic/apyrimidinic sites. Our results
indicate that the preform of human mitochondrial uracil-DNA glycosylase is processed to distinctly different forms lacking
29 or 75/77 N-terminal residues, respectively.
Uracil in DNA results from deamination of cytosine, resulting in mutagenic U : G mispairs, and misincorporation of dUMP, which gives a less harmful U : A pair. At least four different human DNA glycosylases may remove uracil and thus generate an abasic site, which is itself cytotoxic and potentially mutagenic. These enzymes are UNG, SMUG1, TDG and MBD4. The base excision repair process is completed either by a short patch- or long patch pathway, which largely use different proteins. UNG2 is a major nuclear uracil-DNA glycosylase central in removal of misincorporated dUMP in replication foci, but recent evidence also indicates an important role in repair of U : G mispairs and possibly U in single-stranded DNA. SMUG1 has broader specificity than UNG2 and may serve as a relatively efficient backup for UNG in repair of U : G mismatches and single-stranded DNA. TDG and MBD4 may have specialized roles in the repair of U and T in mismatches in CpG contexts. Recently, a role for UNG2, together with activation induced deaminase (AID) which generates uracil, has been demonstrated in immunoglobulin diversification. Studies are now underway to examine whether mice deficient in Ung develop lymphoproliferative malignancies and have a different life span.
Uracil DNA glycosylase hydrolyzes the N-glycosidic bond between sugar phosphate backbone and uracil residue appearing as the result of spontaneous deamination of cytosine or during wrong incorporation of dU residues during DNA synthesis. Uracil DNA glycosylases are very conservative enzymes. They have been recognized in all pro- and eukaryotic organisms and also in pox and herpes viruses. This review highlights the pathways of accumulation of uracil and its derivatives in DNA, the main physicochemical and biochemical properties of uracil DNA glycosylase, and regulation of its functioning. Special attention is paid to detailed mechanisms of recognition and removing of damaged (or wrong) base by uracil DNA glycosylase. These mechanisms have been validated by the methods of X-ray analysis and kinetic and thermodynamic approaches.
Strains of Escherichia coli with a mutation in the sof (dnaS) locus show a higher than normal frequency of recombination (are hyper rec) and incorporate label into short (4-5S) DNA fragments following brief [3H]thymidine pulses [Konrad and Lehman, Proc. Natl. Acad. Sci. USA 72, 2150 (1975)]. These mutant strains have now been found to be defective in deoxyuridinetriphosphate diphosphohydrolase (dUTPase; deoxyuridinetriphosphatase, EC 3.6.1.23), the enzyme that catalyzes the hydrolysis of dUTP to dUMP and PPi. Reversion of one sof- mutation to sof+ restores dUTPase activity and abolishes the accumulation of labeled 4-5S DNA fragments. Mutants initially isolated as defective in dUTPase (dut-) are also hyper rec and show transient accumulation of short DNA fragments. Both the sof and dut mutations are located at 81 min on the E. coli map, closely linked to the pyrE locus. The sof and dut loci thus appear to be identical. A decrease in dUTPase as a consequence of a sof or dut mutation may result in the increased incorporation of uracil into DNA. Rapid removal of the uracil by an excision-repair process could then lead to the transient accumulation of short DNA fragments. It is possible that at least a portion of the Okazaki fragments seen in wild-type cells may originate in this way.
A monoclonal antibody prepared against a partially purified human uracil DNA glycosylase was found, on further purification of the enzyme, to be inactive against the glycosylase. However, immunoreactivity was observed in other protein fractions that contained DNA polymerase activity. The immunoreactive protein was purified to homogeneity and identified as a catalytic subunit of DNA polymerase alpha by molecular mass, by aphidicolin sensitivity, and by recognition by a monoclonal antibody against human KB cell DNA polymerase alpha. Our monoclonal antibody had no effect on homogeneous human uracil DNA glycosylase activity but severely inhibited the activity of the homogeneous human DNA polymerase alpha catalytic subunit. The suspicion that the two proteins were physically associated was confirmed by finding that, on mixing the DNA polymerase alpha subunit with the glycosylase, the latter was strongly inhibited by our monoclonal antibody. These results demonstrate that this monoclonal antibody recognizes not only the DNA polymerase alpha subunit but also the uracil DNA glycosylase when it is physically attached to the polymerase subunit. These results contribute to the definition of relationships between those proteins that may comprise the human base-excision repair multienzyme complex.
A series of monoclonal antibodies has been prepared against the base excision repair enzyme uracil DNA glycosylase isolated from human placenta. Spleen cells from BALB/c mice immunized with purified human placental uracil DNA glycosylase were fused with either P3X63 Ag8.653 or SP2/0 myeloma cells. Hybridomas producing antibodies directed against the placental glycosylase were identified in an enzyme-linked immunosorbent assay. Each positive hybridoma was cloned twice by limit dilution and tested for anti-glycosylase activity in an enzyme immunoprecipitation assay. Each of the four clones examined in detail precipitated enzyme activity in an immunoprecipitation reaction only in the presence of rabbit anti-mouse IgG as a second antibody. No anti-uracil DNA glycosylase activity was observed in a spontaneous hybridoma used as a control. Each monoclonal antibody immunoprecipitated uracil DNA glycosylases isolated from several human tissues. Partial crossreactivity was observed with rat liver glycosylase and with a hamster enzyme. In contrast, no crossreactivity was observed with yeast or Escherichia coli glycosylase. Glycerol gradient sedimentation analysis demonstrated that one of the antibodies bound to the glycosylase at a site that did not diminish its catalytic activity. A second monoclonal antibody bound at a determinant that affected catalytic activity. Analysis of antibody-glycosylase interactions suggests that human cells contain antigenically distinct glycosylase species that may be encoded by individual uracil DNA glycosylase genes. The potential use of these monoclonal antibodies in studies examining the regulation of glycosylase isoenzymes during cell proliferation in normal human cells and in cells from cancer-prone individuals is considered.
Uracil-DNA glycosylase was partially purified from HeLa cells. Various substrates containing [3H]dUMP residues were prepared by nick-translatiqn of calf thymus DNA. The standard substrate was double-stranded DNA with
[3H]dUMP located internally in the chain. Compared to the release of uracil from this substrate, a 3-fold increase in the rate
was seen with single-stranded DNA, and a 20-fold reduction in the rate was observed when the [3H]dUMP-residue was located at the 3′end. The rate of [3H]uracil release decreased progressively when one, two or three of the dNMP residues were replaced by the corresponding rNMP;
in the extreme case when the substrate contained [3H]dUMP in addition to rCMP, rGMP and rAMP, the rate of [3H]uracil release was less than 3% of that of the control. The enzyme was inhibited to the same extent by uracil and the uracil
analogs 6-aminouracil and 5-azauracil, but very weakly, or not at all, by 5 other analogs. Our results suggest strongly that
uracil-DNA glycosylase has a high degree of selectivity for uracil in dUMP residues located internally in DNA chains and that
the recognition of the correct substrate also depends on the residues flanking dUMP being deoxyribonucleotides.
The DNA-agar procedure takes advantage of the fact that complementary single strands of polynueleotides can form duplex structures in vitro under appropriate conditions of incubation. In the technique, high molecular weight single-stranded DNA is immobilized in agar, radioactive single-stranded fragments of DNA or molecules of RNA are allowed to interact with the DNA-agar preparation, and the extent of interaction between the radioactive and the immobilized components is determined. The DNA-agar technique has been used in work with bacteria, animals, plants, virus-host systems, and synthetic polynucleotides. Two methods are convenient for assay of the immobilized single stranded DNA. One involves direct spectrophotometric measurement of the trapped DNA and the other resorts to the release of nucleotide fragments from the DNA-agar after DNase treatment. The chapter also discusses the preparation of DNA-agar, nucleic acid interactions employing DNA-aga, and precautions regarding significant application of the procedures.
The silver-staining procedure for detecting proteins in polyacrylamide gels has been modified and further simplified so that it is stable, controllable, and even more rapid than previous silver-staining methods. The method retains its sensitivity to proteins at the nanogram level and may be used either before or after Coomassie blue staining. Reproducible staining patterns are obtained, and the method is inexpensive, completely under the control of the user, and effective with the common polyacrylamide gel electrophoresis methods.
When dUTP replaced dTTP during polyoma DNA replication in isolated cell nuclei, radioactivity from labeled deoxynucleoside triphosphates was almost exclusively recovered in very short Okazaki fragments and incorporation ceased after a short time. Addition of uracil, a known inhibitor of the enzyme uracil-DNA glycosidase (Lindahl et al., 1977), increased total synthesis and shifted the incorporation to longer progeny strands. The presence of as little as 2.5% of dUTP in a dTTP-containing system gave a distinct increase in isotope incorporation into Okazaki pieces accompanied by a corresponding decrease in longer strands. This effect was reversed completely by uracil. The short strands formed from dUTP could be chased efficiently into long strands. Our results suggest that dUTP can be incorporated in place of dTTP into polyoma DNA, and that polyoma-infected nuclei, similar to E. coli (Tye et al., 1977), contain an excision-repair system which by removal of uracil causes strand breakage and under certain circumstances may contribute to the formation of Okazaki fragments.
[3H]dUMP was incorporated into DNA of isolated S-phase HeLa S3 cell nuclei during DNA synthesis. The incorporated radioactivity was made acid soluble during a chase with excess TTP. A partially purified DNA polymerase alpha incorporated [3H]dUMP into activated salmon sperm DNA. The incorporation rate was equal to the incorporation of [3H]TMP, and the radioactivity incorporated was not made acid soluble during a chase. The nuclei thus have the ability to remove misincorporated uracil. From cytosol we have partially purified an enzyme (80 times purification) that splits the N-glycosidic bond between uracil and deoxyribose in dUMP-containing DNA. This uracil-N-glycosidase has a molecular weight of about 50 000. It does not accept dUTP or RNA as substrates. Pulse labelling of isolated nuclei with radioactive deoxyribonucleoside triphosphates in the presence of dUTP lead to a large accumulation of label in small DNA fragments. The size of these fragments was about 80 nucleotides in a 60 s pulse and no increase in size was observed with increasing pulse length. The corresponding value for control experiments with no dUTP, was 200 nucleotides and the fragments increased in size with increasing pulse length. About 90% of the radioactivity was found in the small fragments after a 3 min pulse when the concentration of dUTP in the test mixture was 100 micrometer and no exogenous TTP was present. In control experiments with no dUTP present, only 14% of the radioactivity was found in small DNA pieces. When test mixture containing dUTP was preincubated with cytosol for 60 s before adding the isolated nuclei, the small fragments increased in size to that of DNA fragments found in control incubations; also the relative amount of label bound to the fragments returned to the levels found in the controls. Increasing the TTP concentration from 5 micrometer to 1.88 mM in the absence of exogenous dUTP had no effect on the size of the DNA fragments.
O-Phthalaldehyde, in the presence of 2-mercaptoethanol, reacts with primary amines to form highly fluorescent products. Picomole quantities of amino acids, peptides, and proteins can be detected easily. o-Phthalaldehyde is five to ten times more sensitive than fluorescamine and is soluble and stable in aqueous buffers.
Uracil-DNA glycosylase, the enzyme that catalyzes the release of free uracil from single-stranded and double-stranded DNA, has been purified 26 600-fold from HeLa S3 cell extracts. The enzyme preparation was essentially homogeneous as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The native enzyme is a small monomeric protein of molecular mass 29 kDa. A minor uracil-DNA glycosylase preparation was also obtained in the final chromatographic step. This preparation is homogeneous with a molecular mass of 29 kDa and may represent the mitochondrial enzyme.
This report also presents a 700-fold purification of HeLa S3 cell O6-methylguanine-DNA methyltransferase. The glycosylase and methyltransferase showed very similar chromatographic properties. The report indicates that the lability of the methyltransferase upon purification may be a consequence of the total separation of the two DNA repair enzymes or of the possibility that some other stabilizing factor is involved.
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.
We have isolated and partially characterized a uracil-DNA glycosylase activity from the cellular slime mold, Dictyostelium discoideum. This glycosylase has a broad pH optimum (6.5-8.5) and is fully active in 10 mM EDTA or in 5 mM Mg2+. Its molecular weight by gel filtration is about 55 000. This enzyme activity may work in concert with previously described apurinic/apyrimidinic (AP) endonuclease activities in the excision repair of uracil from the DNA of this lower eukaryote.
A procedure has been developed which allows the immobilization on glass-fiber sheets coated with the polyquaternary amine, Polybrene, of proteins and protein fragments previously separated on sodium-dodecylsulfate-containing polyacrylamide gels. The transfer is carried out essentially as has been used for protein blotting on nitrocellulose membranes [Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl Acad. Sci. USA 76, 4350-4354], but is now used to determine the amino acid composition and partial sequence of the immobilized proteins. Protein transfer could be carried out after staining the proteins in the gels with Coomassie blue, by which immobilized proteins are visible as blue spots, or without previous staining, after which transferred proteins are detected as fluorescent spots following reaction with fluorescamine. The latter procedure was found to be more efficient and yielded binding capacities of +/- 20 micrograms/cm2. Fluorescamine detection was of equal or higher sensitivity than the classical Coomassie staining of proteins in the gel. Immobilized proteins could be hydrolyzed when still present on the glass fiber and reliable amino acid compositions were obtained for various reference proteins immobilized in less than 100 pmol quantities. In addition, and more importantly, glass-fiber-bound proteins could be subjected to the Edman degradation procedure by simply cutting out the area of the sheet carrying the immobilized protein and mounting the disc in the reaction chamber of the gas-phase sequenator. Results of this immobilization-sequencing technique are shown for immobilized myoglobin (1 nmol) and two proteolytic fragments of actin (+/- 80 pmol each) previously separated on a sodium-dodecylsulfate-containing gel.
Uracil-DNA glycosylase activity in HeLa S3 cells was found in nuclei (70%), mitochondria (15%) and cytosol (15%) after fractionation in hypotonic buffers. After fractionation in isotonic buffers the activity in cytosol was increased, apparently as a consequence of leakage from the nuclei. Both in the nuclear and the mitochondrial fraction, a major 50 and a minor 18 kDa form were found after gel filtration in the presence of 0.5 M NaCl. However, after glycerol, gradient sedimentation or gel filtration in the presence of 2 M NaCl or 20% glycerol most of the 50 kDa form dissociated into a 22 kDa form, which was also the smallest catalytically active form found after partial trypsin digestion. The dissociation of the 50 kDa form was reversible. Biochemical properties of the nuclear and mitochondrial forms were very similar. Thus, they had similar apparent Km values, pH optima, heat sensitivities and activation energies, and were stimulated 2-5-fold by 40-60 mM monovalent salt.
A protein assay is described in which the sample is precipitated with trichloroacetic acid in the presence of sodium dodecylsulfate, filtered off on a Millipore membrane and stained with Amidoschwarz 10B. The proteindye complex is eluted, and its absorbance determined at 630 nm. This assay is very reproducible, insensitive to variations in assay conditions, and linear from 3 to 30 μg of protein. It can be used on samples with a concentration as low as 0.75 μg/ml. There is no interference by commonly used reagents such as Tris, thiol reagents, EDTA, urea, sucrose, and many others. The color yield for a variety of proteins was determined and found to lie within ±15% of the value for bovine serum albumin which was used as standard. Of the proteins tested only insulin, which due to its low molecular weight was incompletely retained on the membrane in the filtration step, gave a low color yield, 50% of the standard.
The rate of deamination of cytosine residues in single-stranded and double-stranded Escherichia coli DNA, in the polynucleotides poly(dC) and poly(dG)·poly(dC), and in dCMP was investigated as a function of temperature, pH, and buffer composition. For this purpose, nucleic acids and polydeoxynucleotides specifically radioactively labeled in the cytosine residues were prepared. After heat treatment, the polymers were enzymatically degraded to mononucleotides or nucleosides, cytosine and uracil derivatives were separated by paper chromatography, and their radioactivity was determined. Cytosine in single-stranded DNA, poly(dC), or dCMP is similarly susceptible to hydrolytic deamination at pH 7.4, and the reaction proceeds at a rate of k = 2 × 10-7 sec-1 at 95°. From measurements at several temperatures it is estimated that the reaction is associated with an activation energy of 29 kcal/mol. These data indicate that a significant amount of conversion of cytosine to uracil occurs during heat denaturation of DNA by standard procedures. The cytosine residues in native DNA are well protected, and are deaminated at <1% of the rate observed with dCMP or poly(dC). In contrast, the cytosine residues in poly(dG)·poly(dC) were deaminated at 75% of the rate of those in poly(dC). The in vivo rate of deamination of cytosine residues in DNA is discussed.
An enzyme that liberates uracil from single-stranded and double-stranded DNA containing deaminated cytosine residues and from deoxycytidylate-deoxyuridylate copolymers in the absence of Mg(++) has been purified 30-fold from cell extracts of E. coli. The enzyme does not release uracil from deoxyuridine, dUMP, uridine, or RNA, nor does it liberate the normally occurring pyrimidine bases, cytosine and thymine, from DNA. The enzymatic cleavage of N-glycosidic bonds in DNA occurs without concomitant cleavage of phosphodiester bonds, resulting in the formation of free uracil and DNA strands of unaltered chain length that contain apyrimidinic sites as reaction products. The enzyme may be active in DNA repair, converting deaminated dCMP residues to an easily repairable form.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
Uracil-DNA glycosylase re_leases free uracil from dUMP-residues in DNA ([l-S], reviewed [6]). Uracil in DNA may arise from misincorporation of dUMP during DNA replication [7-91 or deamination of cytosine in DNA [lo]. Much information is available on the biophysical and biochemical properties [l-6] of uracil-DNA glycosylase, but little is known about how this enzyme is organized in chromatin. Isolated SV40- or polyoma minichromosomes [ 1 l-131 serve as useful models for studies on chromatin proteins. Thus, DNA polymerases (Y and y [13,14-171 as well as T-antigen [ 18,191 are associated with replicating SV40 and/or polyoma minichromosomes, while DNA polymerase 0 and topoisomerase I cosedimented with mature minichromosomes [ 171. Here, we show that uracil-DNA glycosylase, a major DNA repair enzyme, is preferentially Pssociated with replicating SV40 minichromosomes. somes, 0.5 ml extract was layered on a linear 5-30% sucrose gradient in 10 mM Hepes (pH 7.8), 5 mM KC1 and 0.5 mM dithiothreitol [ 1 l] and centrifuged at 37 000 rev./min in a Beckman SW 40 rotor for 150 min at 4°C. Fractions of 0.34 ml were collected from the bottom of the tube. Uracil-DNA glycosylase was tested by incubating aliquots of 30 ~1 from the fractions with 10 ~1 assay buffer (200 mM NaCl, 8 mM EDTA, 160 mM Tris- HCl (pH 7.5) and 12 yM d [3H] UMP-containing DNA, spec. act. 500 /.Li/pmol) for 45 min at 30°C. The release of acid- or ethanol-soluble radioactivity was monitored [5]. The amount of radioactivity released did not exceed 30% of the added radioactivity. The release of [3H]uracil was linear with time (up to 45 min) and with the amount of extract added (up to 30 ~1). Cytochrome c oxidase [22] was determined in aliquots of 100 ~1 from extracts or gradient frac- tions. The detection limit was 0.025 nmol cytochrome c oxidized/min at 25°C. 2.
As a step towards understanding the significance of DNA repair enzymes in the protection against genotoxic and carcinogenic agents, we have examined the activity of O6-methyl-guanine-DNA methyltransferase and uracil-DNA glycosylase in adult human liver, stomach, small intestine and colon. Liver had on average a 5- to 8-fold higher activity of O6-MeG-DNA methyltransferase than the other organs and showed about an 8-fold inter-individual variation. In colon and small intestine an even larger inter-individual variation was observed (10- and 40-fold, respectively). In two colon tumors examined the activity of O6-MeG-DNA methyltransferase was several fold higher than in non-neoplastic colon mucosa from the same individuals, while uracil-DNA glycosylase activity was essentially equal in neoplastic and non-neoplastic tissues. O6-MeG-DNA methyltransferase activities in two gastric tumors examined were not higher than in average non-neoplastic tissue. In general the activity of uracil-DNA glycosylase did not correlate with the O6-MeG-DNA methyltransferase activity. The inter-individual variation of this enzyme in the activity was only 3-fold in liver and normal stomach, but varied 5.5 and 60-fold in colon and small intestine, respectively. In conclusion, we have found that O6-MeG-DNA methyltransferase as well as uracil-DNA glycosylase activity vary considerably between different tissues as well as between different individuals. Whether this variation has a genetic basis or reflects variation in 'life style' is not known.
The activities of the DNA repair enzymes O6-methylguanine-DNA methyltransferase and uracil-DNA glycosylase, and the replicative enzyme DNA polymerase α, were measured
in extracts of human fetal tissues at 18–20 weeks of gestation. In general, O6-methylguanine-DNA methyltransferase activities in fetal tissues were in the same range as in the corresponding adult tissues,
except for fetal liver which had ∼ 5-fold lower activity. Uracil-DNA glycosylase was, surprisingly, ∼4-fold lower in fetal
tissues compared with adult tissues. Since a critical factor in carcinogenesis may be the rate of repair relative to DNA replication,
the activities of O6-methyl-guanine-DNA methyltransferase and uracil-DNA glycosylase were compared with the DNA polymerase α activity in the same
extract. When expressed in this way, O6-methylguanine-DNA methyltransferase activity was lowest in liver and brain and 2- to 14-fold higher in kidney, lung, colon,
stomach, small intestine and pancreas. The ratio of uracil-DNA glycosylase to DNA polymerase αvaried less between different
organs. These findings indicate that several fetal organs may be more sensitive than adult organs to some alkylating agents
that are known to occur in the environment. Furthermore, the lower capacity of DNA repair is not restricted to repair of alkylation
damage, since the activity of uracil-DNA glycosylase is also lower than in adult tissues.