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Processivity of uracil DNA glycosylase
Abstract
The purpose of this study was to determine the mechanism by which uracil DNA glycosylase locates uracil residues within double-stranded DNA. Using reaction conditions that contained low salt concentrations, the addition of uracil DNA glycosylase to plasmid DNAs containing multiple, randomly incorporated uracils resulted in the accumulation of form III DNA while unreacted form I DNA was still present. These data suggested that the enzyme utilizes a one-dimensional DNA-scanning mechanism such that this linear DNA arose by the accumulation of many single-strand breaks within the plasmid prior to enzyme dissociation. Reactions containing higher concentrations of uracil DNA glycosylase revealed a further accumulation of form III DNA after all form I DNA had been lost. These results suggested a partial (1.5-2 kb) enzyme processivity since the enzyme does not incise at all uracil bases on the DNA molecule prior to dissociation from that DNA. Since DNA scanning is regulated by electrostatic interactions, the processivity of the enzyme was evaluated through kinetic analyses of incision at various salt concentrations. At NaCl concentrations (< 50 mM), a significant amount of form III DNA accumulated while there were still unreacted form I DNAs present. In contrast, the accumulation of form III DNA was delayed at higher salt concentrations and the overall accumulation of form III DNA was less than that monitored at lower salt concentrations. DNAs were also analyzed by denaturing agarose gel electrophoresis in order to measure the average distance between strand breaks. Southern hybridizations showed a greater accumulation of breaks in DNAs that were reacted with the uracil DNA glycosylase at the lower salt concentrations, confirming a partial processivity for the enzyme.
... The kinetics of the enzyme from E.coli have been thoroughly studied both on single and double stranded DNA and various oligonucleotides (Lindahl et al., 1977;Varshney and van de Sande, 1990;Eftedal et al., 1993). This enzyme has been shown to bind DNA in a non-specific manner and act processively with an average scan length of 1.5 to 2 kilobases before dissociation (Higley and Lloyd, 1993). In an investigation carried out on the kinetics of uracil excision from oligonucleotides by the E.coli enzyme, it was noted that the shortest substrate is a dimeric oligonucleotide phosphorylated at both ends with the uracil at the 5' end, or a trinucleotide phosphorylated at the 5' end only, with the uracil at the 5' end (Varshney and van de Sande, 1990). ...
... UDGases have been shown to bind to single or double stranded DNA in a non specific manner (Higley and Lloyd, 1993), and to short oligomers of DNA (Varshney and van de Sande, 1991), also to be inhibited by uracil -the end product of the reaction, and to an extent by certain uracil analogs (Krokan and Wittwer, 1981). It was hoped that co crystallisation of an enzyme/ DNA or en2yme/ inhibitor complex could be achieved. ...
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.
... These experiments measure the rate at which a glycosylase cleaves consecutive damage sites within a single DNA strand of a duplex. The processivity of E. coli uracil DNA glycosylase (Udg) was examined using both plasmid [21] and linear DNA substrates [22][23][24][25]. Similar to what was observed for T4 endonuclease V, the processivity of Udg appears to be highly dependent on salt conditions for both substrates. ...
... Similar to what was observed for T4 endonuclease V, the processivity of Udg appears to be highly dependent on salt conditions for both substrates. However, Udg also appears to switch between a distributive and processive mechanism depending on the identity of the linear substrate [21][22][23][24]26]. As evidence for a combination of sliding and distributive search modes grew, linear substrates were designed with precisely-spaced damage sites to allow for a determination of how far the glycosylase slides before dissociating from the DNA strand. ...
The first step of base excision repair utilizes glycosylase enzymes to find damage within a genome. A persistent question in the field of DNA repair is how glycosylases interact with DNA to specifically find and excise target damaged bases with high efficiency and specificity. Ensemble studies have indicated that glycosylase enzymes rely upon both sliding and distributive modes of search, but ensemble methods are limited in their ability to directly observe these modes. Here we review insights into glycosylase scanning behavior gathered through single-molecule fluorescence studies of enzyme interactions with DNA and provide a context for these results in relation to ensemble experiments.
... Processive enzymes differ greatly in the " extent " of their processivity, i.e., in the average number of subunits covered before dissociation (Table 1). Some enzymes, like restriction endonucleases and DNA-repair enzymes, are considered quasiprocessive, as they are able to slide along 200 (Carey and Strauss 1999) to 2000 (Higley and Lloyd 1993) base pairs. At the other extreme, certain polymerase holoenzymes , responsible for replicating genetic material, seem virtually unlimited in this respect. ...
... Several examples of partially enclosing processive enzymes that bind DNA are shown in Figure 1. An interesting example of this class is uracil DNA glycosylase (UDG) (Fig. 1a), responsible for processively scanning and removing misincorporated uracil bases from DNA (Higley and Lloyd 1993). Cocrystal structures of UDG with DNA that contains uracil bases show that a strand from the double helix of DNA is bound in a cleft on the surface of UDG (Parikh et al. 1998). ...
The structures of a number of processive enzymes have been determined recently. These proteins remain attached to their polymeric substrates and may perform thousands of rounds of catalysis before dissociating. Based on the degree of enclosure of the substrate, the structures fall into two broad categories. In one group, the substrate is partially enclosed, while in the other class, enclosure is complete. In the latter case, enclosure is achieved by way of an asymmetric structure for some enzymes while others use a symmetrical toroid. In those cases where the protein completely encloses its polymeric substrate, the two are topologically linked and an immediate explanation for processivity is provided. In cases where there is only partial enclosure, the structural basis for processivity is less obvious. There are, for example, pairs of proteins that have quite similar structures but differ substantially in their processivity. It does appear, however, that the enzymes that are processive tend to be those that more completely enclose their substrates. In general terms, proteins that do not use topological restraint appear to achieve processivity by using a large interaction surface. This allows the enzyme to bind with moderate affinity at a multitude of adjacent sites distributed along its polymeric substrate. At the same time, the use of a large interaction surface minimizes the possibility that the enzyme might bind at a small number of sites with much higher affinity, which would interfere with sliding. Proteins that can both slide along a polymeric substrate, and, as well, recognize highly specific sites (e.g., some site-specific DNA-binding proteins) appear to undergo a conformational change between the cognate and noncognate-binding modes.
... Sliding along the DNA backbone without release is often referred to as "facilitated diffusion," but glycosylases may also make small "hops" between close sites on the DNA or traverse longer distances through intrastrand and/or interstrand transfer events (for reviews see [54][55][56]). Ensemble methods measuring correlated cleavage of closely spaced damage sites in long oligodeoxyribonucleotides provided evidence that the glycosylases were capable of remaining in continuous contact with the DNA molecule after removing a lesion [57][58][59][60][61][62][63][64]. However, direct visualization of the movement of glycosylases along longer tracts of undamaged DNA remained a challenge until a breakthrough were several thousand bases in length. ...
The first step of the base excision repair (BER) pathway responsible for removing oxidative DNA damage utilizes DNA glycosylases to find and remove the damaged DNA base. How glycosylases find the damaged base amidst a sea of undamaged bases has long been a question in the BER field. Single molecule total internal reflection fluorescence microscopy (SM TIRFM) experiments have allowed for an exciting look into this search mechanism and have found that DNA glycosylases scan along the DNA backbone in a bidirectional and random fashion. By comparing the search behavior of bacterial glycosylases from different structural families and with varying substrate specificities, it was found that glycosylases search for damage by periodically inserting a wedge residue into the DNA stack as they redundantly search tracks of DNA that are 450–600 bp in length. These studies open up a wealth of possibilities for further study in real time of the interactions between DNA glycosylases and other BER enzymes with various DNA substrates.
... The processive sliding mechanism has been tested using correlated cleavage experiments, and it does appear that several glycosylases are able to sequentially process closely spaced lesions. However, these assays are unable to directly observe long range interactions between the glycosylase and DNA, and they also are unable to characterize glycosylase interactions with undamaged DNA (Bennett et al., 1995;Dowd and Lloyd, 1990;Gruskin and Lloyd, 1988;Higley and Lloyd, 1993;Mechetin and Zharkov, 2011;Porecha and Stivers, 2008;Purmal et al., 1994;Sidorenko et al., 2008). ...
The Base Excision Repair (BER) pathway removes the vast majority of damages produced by ionizing radiation, including the plethora of radiation-damaged purines and pyrimidines. The first enzymes in the BER pathway are DNA glycosylases, which are responsible for finding and removing the damaged base. Although much is known about the biochemistry of DNA glycosylases, how these enzymes locate their specific damage substrates among an excess of undamaged bases has long remained a mystery. Here we describe the use of single molecule fluorescence to observe the bacterial DNA glycosylases, Nth, Fpg and Nei, scanning along undamaged and damaged DNA. We show that all three enzymes randomly diffuse on the DNA molecule and employ a wedge residue to search for and locate damage. The search behavior of the Escherichia coli DNA glycosylases likely provides a paradigm for their homologous mammalian counterparts.
... The second assay uses a concatemer substrate ligated from a number of identical dsODN units, with the processive cleavage evident as preferential accumulation of unit-length products [79]. Eco-Ung has been studied using both the plasmid assay [80] and the concatemer assay [79,81], with the former one demonstrating processivity over the range of 1.5-2 kb and the latter one suggesting either the distributive [79] or processive [81] mode of action. Rat liver mitochondrial UNG was reported to be processive in the concatemer assay [81]. ...
Uracil appears in DNA as a result of cytosine deamination and by incorporation from the dUTP pool. As potentially mutagenic and deleterious for cell regulation, uracil must be removed from DNA. The major pathway of its repair is initiated by uracil-DNA glycosylases (UNG), ubiquitously found enzymes that hydrolyze the N-glycosidic bond of deoxyuridine in DNA. This review describes the current understanding of the mechanism of uracil search and recognition by UNG. The structure of UNG proteins from several species has been solved, revealing a specific uracil-binding pocket located in a DNA-binding groove. DNA in the complex with UNG is highly distorted to allow the extrahelical recognition of uracil. Thermodynamic studies suggest that UNG binds with appreciable affinity to any DNA, mainly due to the interactions with the charged backbone. The increase in the affinity for damaged DNA is insufficient to account for the exquisite specificity of UNG for uracil. This specificity is likely to result from multistep lesion recognition process, in which normal bases are rejected at one or several pre-excision stages of enzyme-substrate complex isomerization, and only uracil can proceed to enter the active site in a catalytically competent conformation. Search for the lesion by UNG involves random sliding along DNA alternating with dissociation-association events and partial eversion of undamaged bases for initial sampling.
... Common attributes are that the enzyme has a starting point, the primer/template junction, and then sequentially moves in one direction inserting nucleotides opposite template DNA. Numerous other enzymes use three-dimensional movement by facilitated diffusion, such as DNA glycosylases (Higley & Lloyd 1993;Bennett et al. 1995), human AP endonuclease (Carey & Strauss 1999), T4 endonuclease V (Dowd & Lloyd 1990), restriction endonucleases (Jack et al. 1982;Terry et al. 1985;Stanford et al. 2000) and AID and APOBEC3G (Pham et al. 2003(Pham et al. , 2007Chelico et al. 2006). These enzymes face a formidable challenge of finding 'needle in a haystack' substrate motifs in DNA after an arbitrary starting point is established by an initial random binding event. ...
Activation-induced (cytidine) deaminase (AID) efficiently introduces multiple and diversified deaminations in immunoglobulin (Ig) variable and switch regions. Here, we review studies of AID, and the APOBEC family member, APOBEC3G, demonstrating that both enzymes introduce multiple deaminations by processive action on single-stranded DNA and that deaminations occur stochastically at hot- and cold-spot targets. In a more detailed analysis of AID, we examine phosphorylation-null mutants, particularly, S38A and S43P. S43P mutant AID has been identified in a patient with hyper-IgM immunodeficiency syndrome. The phosphorylation-null mutants have essentially the same specific activity, processivity and ability to undergo transcription-dependent deamination compared with wild-type (WT) AID. Although the phosphorylation-null mutants still deaminate 5'-WRC hot spots, the mutant deamination spectra differ from WT AID. The mutants strongly prefer two motifs, 5'AGC and 5'GGC, which are disfavoured by WT AID. Differences in deamination specificities can be attributed primarily to the replacement of Ser rather than to the absence of phosphorylation. The 5'GGC motif occurs with exceptionally high frequency on the non-transcribed strand of human switch regions, IgG4 and IgE. The potential for S43P to catalyse large numbers of aberrant deaminations in switch region sequences suggests a possible relationship between non-canonical AID deamination specificity and a loss of antibody diversification.
... Linear diffusion requires non-specific binding to DNA followed by moving along the DNA in a search for specific sites (Berg et al., 1981; Hanawalt, 1993). Among the DNA repair enzymes, uracil DNA-glycosylase and T4 endoV are processive in low salt conditions, but distributive in higher salt (Ganesan et al., 1986; Gruskin and Lloyd, 1986; Higley and Lloyd, 1993). On naked DNA, NER is achieved via a random diffusion mechanism (Szymkowski et al., 1993), but one-dimen-sional search was proposed for Rad7–Rad16 (Guzder et al., 1998b) and for photolyase in low salt (van Noort et al., 1998 ). ...
Nucleotide excision repair (NER) and DNA repair by photolyase in the presence of light (photoreactivation) are the major pathways to remove UV-induced DNA lesions from the genome, thereby preventing mutagenesis and cell death. Photoreactivation was found in many prokaryotic and eukaryotic organisms, but not in mammals, while NER seems to be universally distributed. Since packaging of eukaryotic DNA in nucleosomes and higher order chromatin structures affects DNA structure and accessibility, damage formation and repair are coupled intimately to structural and dynamic properties of chromatin. Here, I review recent progress in the study of repair of chromatin and transcribed genes. Photoreactivation and NER are discussed as examples of how an individual enzyme and a complex repair pathway, respectively, access DNA lesions in chromatin and how these two repair processes fulfil complementary roles in removal of UV lesions. These repair pathways provide insight into the structural and dynamic properties of chromatin and suggest how other DNA repair processes could work in chromatin.
... Interestingly, the NaCl-optimum for cUNG increases as pH decreases. It has previously been demonstrated that UDG functions in a processive manner at low ionic strength, which, as NaCl concentration increases the enzyme, switches to a distributive mechanism (Higley and Lloyd, 1993; Bennett et al., 1995 ). However, Purmal et al. (1994) reported that UDG acted in a distributive mechanism at low ionic strength. ...
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.
... The interactions between DNA-interacting enzymes and DNA are basically electrostatic in nature, and many of the enzymes operate in a processive manner (Dodson et al. 1994; Lohman 1986; von Hippel and Berg 1989). UNG has been shown to operate in both a processive and distributive manner (Bennett et al. 1995; Higley and Lloyd 1993; Purmal et al. 1994 ), and processivity is probably dependent on a certain pH and NaCl concentration for proper binding to the DNA. The different optima, with respect to pH and NaCl concentration, between the cod and human enzymes could reflect a variation in their electrostatic surface potentials, giving them different DNA-binding characteristics. ...
Two distinct forms of the highly conserved uracil-DNA glycosylase (UNG) have been isolated from Atlantic cod (Gadus morhua) liver cDNA by rapid amplification of cDNA ends (RACE). From the cDNA sequences, both forms were deduced to encode an open reading frame of 301 amino acids, with an identical 267-amino-acid C-terminal region and different N-terminal regions of 34 amino acids. By comparison with the human UNG sequences, the two forms were identified as possible mitochondrial (cUNG1) and nuclear (cUNG2) forms. Several constructs of recombinant cUNG (rcUNG) were expressed in Escherichia coli in order to optimize the yield. The recombinant enzyme was purified to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Activity and stability experiments showed that rcUNG was similar to cUNG previously purified from Atlantic cod liver, and was more pH- and temperature labile than a recombinant human UNG (rhUNG). Under optimal assay conditions for both rcUNG and rhUNG, the turnover number (k(cat)) was three times higher for rcUNG compared with rhUNG, with an identical K(M), resulting in a threefold higher catalytic efficiency (k(cat)/K(M)) for rcUNG. These activity and stability experiments reveal cold-adapted features in rcUNG. Homology models of the catalytic domains of Atlantic cod (cUNG) and mouse uracil-DNA glycosylase (mUNG) were built using the human UNG (hUNG) crystal structure as a template. The unique amino acid substitutions observed in cod UNG were mainly located in the N- and C-terminal parts of the sequence. The analysis indicated a more stable N-terminal, a more flexible C-terminal, and a less stabilized core in cUNG as compared with the mammalian UNGs. Substitution of several amino acids in or near the DNA-binding site in cUNG could give rise to a more positively charged surface and a higher electrostatic potential near the active site compared with the mammalian UNGs. The higher potential may increase the electrostatic interactions between the enzyme and DNA, and may explain the increased substrate affinity and, in combination with the higher flexibility, the higher catalytic efficiency observed for rcUNG.
To store the genetic information and serve as the genetic link between generations, the nucleotide sequence of DNA must be faithfully maintained despite the numerous physical or chemical insults discussed in the previous chapter. To that end, all organisms from bacteria to mammals are endowed with DNA repair mechanisms, and even some viral genomes carry their own DNA repair enzymes.
To perform their functions, many DNA-dependent proteins have to quickly locate specific targets against the vast excess of nonspecific DNA. Although this problem was first formulated over 40 years ago, the mechanism of such search remains one of the unsolved fundamental problems in the field of protein-DNA interactions. Several complementary mechanisms have been suggested: sliding, based on one-dimensional random diffusion along the DNA contour; hopping, in which the protein "jumps" between the closely located DNA fragments; macroscopic association-dissociation of the protein-DNA complex; and intersegmental transfer. This review covers the modern state of the problem of target DNA search, theoretical descriptions, and methods of research at the macroscopic (molecule ensembles) and microscopic (individual molecules) levels. Almost all studied DNA-dependent proteins search for specific targets by combined three-dimensional diffusion and one-dimensional diffusion along the DNA contour.
Translocation of carbohydrate polymers through protein tunnels and clefts is a ubiquitous biochemical phenomenon in proteins such as polysaccharide synthases, glycoside hydrolases, and carbohydrate-binding modules. Although static snapshots of carbohydrate polymer binding in proteins have long been studied via crystallography and spectroscopy, the molecular details of polysaccharide chain processivity have not been elucidated. Here, we employ simulation to examine how a cellulose chain translocates by a disaccharide unit during the processive cycle of a Glycoside Hydrolase Family 7 cellobiohydrolase. Our results demonstrate that these biologically and industrially important enzymes employ a two-step mechanism for chain threading to form a Michaelis complex, and that the free energy barrier to chain threading is significantly lower than the hydrolysis barrier. Taken with previous studies, our findings suggest that the rate-limiting step in enzymatic cellulose degradation is the glycosylation reaction, not chain processivity. Based on the simulations, we find that strong electrostatic interac-tions with polar residues that are conserved in GH7 cellobiohydrolases, but not in GH7 endoglucanases, at the leading glucosyl ring provide the thermodynamic driving force for polysaccharide chain translocation. Also, we consider the role of aromatic-carbohydrate interactions, which are widespread in carbohydrate-active enzymes and have long been associated with processivity. Our analysis suggests that the primary role for these aromatic residues is to provide tunnel shape and guide the carbohydrate chain to the active site. More broadly, this work elucidates the role of common protein motifs found in carbohydrate-active enzymes that synthesize or depolymerize polysaccharides by chain translocation mechanisms coupled to catalysis.
Our cellular genome is continuously exposed to a wide spectrum of exogenous and endogenous DNA damaging agents. These agents can lead to formation of an extensive array of DNA lesions including single- and double-stranded breaks, inter- and intrastrand cross-links, abasic sites, and modification of DNA nucleobases. Persistence of these DNA lesions can be both mutagenic and cytotoxic, and can cause altered gene expression and cellular apoptosis leading to aging, cancer, and various neurological disorders. To combat the deleterious effects of DNA lesions, cells have a variety of DNA repair pathways responsible for restoring damaged DNA to its canonical form. Here we examine one of those repair pathways, the base excision repair (BER) pathway, a highly regulated network of enzymes responsible for repair of modified nucleobase and abasic site lesions.
According to the currently accepted model, enzymes searching for specific recognition sequences or structural elements (modified nucleotides, breaks, single-stranded DNA fragments, etc.) slide at a high rate along DNA. Such sliding is possible only if the enzymes possess sufficiently high affinity for all DNA, sequence notwithstanding. Therefore, significant differences in their affinity for specific and nonspecific DNA sequences are unlikely, and the formation of a complex between an enzyme and its target DNA is not a basic factor of enzyme specificity. To elucidate such factors, we have analyzed many DNA replication, DNA repair, topoisomerization, integration, and recombination enzymes using a number of physicochemical methods, including the method of stepwise increase in ligand complexity developed in our laboratory. It has been shown that high affinity of all studied enzymes for long DNAs is provided by the formation of many weak contacts of the enzyme with all nucleotide units covered by the protein globule. The main role lies in the contact between positively charged amino acid residues and internucleoside phosphate groups; however, the contribution of each contact is very small, and the full contact interface usually resembles that characteristic of interactions between oppositely charged biopolymer surfaces. In some cases, a significant contribution to the affinity is made through hydrophobic and/or van der Waals interactions of the enzymes with nucleotide bases. On the whole, such nonspecific interactions provide for five to eight orders of enzyme affinity for DNA, depending on the enzyme. Specific interactions of enzymes with long DNAs, in contrast to their contacts with small ligands, are usually weak and comparable in efficiency with weak nonspecific contacts. The sum of specific interactions most often provides for approximately one or, rarely, two orders of affinity. According to structural data, DNA binding to any of the investigated enzymes is followed by a stage of DNA conformation adjustment, which includes partial or complete DNA melting, deformation of its backbone, stretching, compression, bending or kinking, eversion of nucleotides from the DNA helix, etc. The full set of such changes is specific for each individual enzyme. The fact that all enzyme-dependent changes in DNA are effected through weak specific (rather than strong) interactions is very important. Enzyme-specific changes in DNA conformation are required for effective adjustment of reacting orbitals to an accuracy of 10–15, which is possible only in the case of specific DNAs. A transition from nonspecific to specific DNA leads to an increase in the reaction rate (k
cat) by four to eight orders of magnitude. Thus, the stages of DNA conformation adjustment and catalysis proper provide for the high specificity of enzyme action.
Uracil-DNA glycosylase (UDG) functions as a sentry guarding against uracil in DNA. UDG initiates DNA base excision repair (BER) by hydrolyzing the uracil base from the deoxyribose. As one of the best studied DNA glycosylases, a coherent and complete functional mechanism is emerging that combines structural and biochemical results. This functional mechanism addresses the detection of uracil bases within a vast excess of normal DNA, the features of the enzyme that drive catalysis, and coordination of UDG with later steps of BER while preventing the release of toxic intermediates. Many of the solutions that UDG has evolved to overcome the challenges of policing the genome are shared by other DNA glycosylases and DNA repair enzymes, and thus appear to be general.
Cells have evolved distinct mechanisms for both preventing and removing mutagenic and lethal DNA damage. Structural and biochemical characterization of key enzymes that function in DNA repair pathways are illuminating the biological and chemical mechanisms that govern initial lesion detection, recognition, and excision repair of damaged DNA. These results are beginning to reveal a higher level of DNA repair coordination that ensures the faithful repair of damaged DNA. Enzyme-induced DNA distortions allow for the specific recognition of distinct extrahelical lesions, as well as tight binding to cleaved products, which has implications for the ordered transfer of unstable DNA repair intermediates between enzymes during base excision repair.
This review will present a current understanding of mechanisms for the initiation of base excision repair (BER) of oxidatively-induced DNA damage and the biological consequences of deficiencies in these enzymes in mouse model systems and human populations.
Uracil-DNA glycosylase (Ung) is a DNA repair enzyme that excises uracil bases from DNA, where they appear through deamination of cytosine or incorporation from a cellular dUTP pool. DNA repair enzymes often use one-dimensional diffusion along DNA to accelerate target search; however, this mechanism remains poorly investigated mechanistically. We used oligonucleotide substrates containing two uracil residues in defined positions to characterize one-dimensional search of DNA by Escherichia coli Ung. Mg(2+) ions suppressed the search in double-stranded DNA to a higher extent than K(+) likely due to tight binding of Mg(2+) to DNA phosphates. Ung was able to efficiently overcome short single-stranded gaps within double-stranded DNA. Varying the distance between the lesions and fitting the data to a theoretical model of DNA random walk, we estimated the characteristic one-dimensional search distance of ~100 nucleotides and translocation rate constant of ~2×10(6) s(-1).
X-ray structural analysis provides no quantitative estimate of the relative contribution of specific and nonspecific or strong and weak interactions to the total affinity of enzymes for nucleic acids. We have shown that the interaction between enzymes and long nucleic acids at the molecular level can be successfully analyzed by the method of stepwise increase in ligand complexity (SILC). In the present review we summarize our studies of human uracil DNA glycosylase and apurinic/apyrimidinic endonuclease, E. coli 8-oxoguanine DNA glycosylase and RecA protein using the SILC approach. The relative contribution of structural (X-ray analysis data), thermodynamic, and catalytic factors to the discrimination of specific and nonspecific DNA by these enzymes at the stages of complex formation, the following changes in DNA and enzyme conformations and especially the catalysis of the reactions is discussed.
Specific and nonspecific DNA complex formation with human uracil-DNA glycosylase, 8-oxoguanine-DNA glycosylase, and apurine/apyrimidine endonuclease, as well as with E. coli 8-oxoguanine-DNA glycosylase and RecA protein was analyzed using the method of stepwise increase in DNA-ligand complexity. It is shown that high affinity of these enzymes to any DNA (10−4–10−8 M) is provided by a large number of weak additive contacts mainly with DNA internucleoside phosphate groups and in a less degree with bases of nucleotide links “covered” by protein globules. Enzyme interactions with specific DNA links are comparable in efficiency with weak unspecific contacts and provide only for one-two orders of affinity (10−1–10−2 M), but these contacts are extremely important at stages of DNA and enzyme structural adaptation and catalysis proper. Only in the case of specific DNA individual for each enzyme alterations in DNA structure provide for efficient adjustment of reacting enzyme atoms and DNA orbitals with accuracy up to 10–15° and, as a result, for high reaction rate. Upon transition from nonspecific to specific DNA, reaction rate (k
cat) increases by 4–8 orders of magnitude. Thus, stages of DNA and enzyme structural adaptation as well as catalysis proper are the basis of specificity of repair enzymes.
Genomic DNA is constantly subjected to damaging modifications from reactive metabolites and environmental mutagens. The base excision repair pathway (BER) is responsible for repair of most modifications affecting single bases, collectively referred to as base lesions. Base lesions are the most frequently occurring type of DNA damage, with ~10,000 base lesions formed and repaired by BER in human cells everyday. However, in the context of the human genome, these base lesions are extremely rare, with only one of every 1.2 million nucleotides sustaining damage on any given day. The daunting task of locating single base lesions and initiating the BER pathway is bestowed upon DNA glycosylases, which catalyze removal of a wide variety of damaged nucleobases from DNA. We show that alkyladenine DNA glycosylase (AAG), a human protein that initiates repair of a diverse group of alkylated and deaminated purines, locates damage by a correlated searching mechanism whereby each binding encounter with DNA involves a search of multiple adjacent sites. This search is mediated by electrostatic binding interactions that allow linear diffusion along nonspecific DNA to locate target sites. We show that the N-terminus of AAG, which is dispensable for glycosylase activity, contributes to the correlated search by decreasing dissociation from DNA, possibly by directly contacting DNA. Furthermore, we demonstrate that AAG makes significant excursions from the surface of DNA while diffusing. Such events, referred to as hops, allow reorientation between strands, enabling AAG to search both strands of a DNA duplex in a single binding encounter. Hopping also allows AAG to bypass obstacles, such as tightly-bound proteins and helix-discontinuities. By comparing the behavior of AAG on oligonucleotides containing different lesions, we show that the efficiency of the search for damage depends on the identity of the base lesion. Whereas AAG recognizes the alkylated lesion, 1, N6-ethenoadenine with high efficiency, AAG requires multiple encounters with the oxidative lesion inosine. We infer that a highly redundant search allows multiple encounters with each lesion, ensuring that each lesion is repaired. Collectively, these studies provide insight into the molecular mechanism by which a DNA repair enzyme searches the genome for DNA damage.
A fundamental and shared process in all forms of life is the use of DNA glycosylase enzymes to excise rare damaged bases from genomic DNA. Without such enzymes, the highly ordered primary sequences of genes would rapidly deteriorate. Recent structural and biophysical studies are beginning to reveal a fascinating multistep mechanism for damaged base detection that begins with short-range sliding of the glycosylase along the DNA chain in a distinct conformation we call the search complex (SC). Sliding is frequently punctuated by the formation of a transient "interrogation" complex (IC) where the enzyme extrahelically inspects both normal and damaged bases in an exosite pocket that is distant from the active site. When normal bases are presented in the exosite, the IC rapidly collapses back to the SC, while a damaged base will efficiently partition forward into the active site to form the catalytically competent excision complex (EC). Here we review the unique problems associated with enzymatic detection of rare damaged DNA bases in the genome and emphasize how each complex must have specific dynamic properties that are tuned to optimize the rate and efficiency of damage site location.
Alkyladenine DNA glycosylase (AAG) initiates the base excision repair pathway that repairs damage to single bases within DNA. AAG recognizes lesions caused by alkylation and deamination. AAG first locates the site of damage then excises the damaged base, leaving an abasic site in the DNA that is further processed by other repair proteins to complete the pathway. Although many substrates have been identified and there are high resolution structures, our understanding of the AAG mechanism remains incomplete. To investigate this further, the thermodynamics and kinetics of binding and base-flipping, along with structural conformational changes of AAG, were investigated. The thermodynamics of AAG binding to damaged and undamaged DNA was examined using fluorescence anisotropy. Surprisingly, this revealed that multiple proteins could bind with nanomolar affinity to short DNA oligonucleotides, which might be a common phenomenon for DNA repair enzymes. These results reveal the pitfalls of studying DNA binding by fluorescence anisotropy, since nonspecific binding dominates the changes in signal. The kinetic mechanism of the AAG reaction with 1,N6-ethenoadenine (??A)-containing DNA was established, including binding, nucleotide flipping, base excision, and product release steps, by taking advantage of the natural fluorescence of the ??A lesion. We observed that the flipping step is fast and the equilibrium for flipping is highly favorable. This kinetic mechanism maximizes specificity between damaged and undamaged bases. To study possible conformational changes in AAG, we took two approaches. First, tyrosine residues in the active site pocket were mutated to tryptophans
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to serve as fluorescence reporters. We found Y127W and Y159W mutants had robust activity towards ??A. However, a full kinetic characterization revealed that these mutations have large effects on the rates and equilibria for flipping. This suggests these mutants will have limited utility in studying recognition and flipping of other damaged nucleotides. Secondly, preliminary experiments established the feasibility of using NMR to study AAG and provided evidence for extensive conformational changes that take place upon binding to DNA. These studies have provided a mechanistic framework that will facilitate future investigations into the role of conserved residues and the energetic basis for the discrimination between damaged and undamaged DNA.
Spontaneous DNA damage occurs throughout the genome, requiring that DNA repair enzymes search each nucleotide every cell cycle. This search is postulated to be more efficient if the enzyme can diffuse along the DNA, but our understanding of this process is incomplete. A key distinction between mechanisms of diffusion is whether the protein maintains continuous contact (sliding) or whether it undergoes microscopic dissociation (hopping). We describe a simple chemical assay to detect the ability of a DNA modifying enzyme to hop and have applied it to human alkyladenine DNA glycosylase (AAG), a monomeric enzyme that initiates repair of alkylated and deaminated purine bases. Our results indicate that AAG uses hopping to effectively search both strands of a DNA duplex in a single binding encounter. This raised the possibility that AAG might be capable of circumnavigating blocks such as tightly bound proteins. We tested this hypothesis by binding an EcoRI endonuclease dimer between two sites of DNA damage and measuring the ability of AAG to act at both damaged sites in a single binding encounter. Remarkably, AAG bypasses this roadblock in approximately 50% of the binding events. We infer that AAG makes significant excursions from the surface of the DNA, allowing reorientation between strands and the bypass of a bound protein. This has important biological implications for the search for DNA damage because eukaryotic DNA is replete with proteins and only transiently accessible.
ABSTRACT: Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy KINETIC CHARACTERIZATION OF SITE-DIRECTED MUTANTS OF THE CONSERVED ACTIVE-SITE PHENYLALANINE OF URACIL-DNA GLYCOSYLASE FROM Escherichia coli By Ryan W. Shaw December 2003 Chair: Linda B. Bloom Major Department: Biochemistry and Molecular Biology Uracil is removed from DNA by the highly conserved uracil-DNA glycosylase (UDG), which hydrolyzes the N-glycosidic bond of deoxyuridine (dU), starting the base excision and repair pathway. This specific and powerful catalyst can lower the activation barrier of deoxyuridine (dU) cleavage by almost 16 kcal/mol, yet UDG exhibits no detectable N-glycosidic bond cleavage of other structurally similar bases. ABSTRACT: In an effort to better understand how UDG is such a powerful and specific catalyst, mutational analysis was performed on a conserved active-site phenylalanine (Phe77) that ?-stacks with the uracil ring of the substrate. The Phe77 mutations did not change the specificity of UDG as the mutants showed no detectable activity toward normal DNA. Under single-turnover conditions the F77A and F77Y mutations had a mild 2.0 and 1.6 kcal/mol destabilizing effects (respectively) on the activation barrier of glycosidic bond cleavage, while changing Phe77 into asparagine has a surprisingly large 4.2 kcal/mol destabilizing effect. The alanine mutation was relatively more detrimental on UDG?s activity toward dsDNA rather than ssDNA substrates, indicating Phe77 contributes to dU binding in duplex DNA. The glycosylase?s ability to bind dU in double-stranded DNA was monitored through changes in its intrinsic tryptophan (Trp) fluorescence. ABSTRACT: As wild-type UDG binds dU-containing DNA its Trp fluorescence quenches and then recovers as the enzyme releases the products after catalysis. The F77N and F77Y mutations had little effect on the Trp quenching kinetics, while the F77A mutant?s Trp signal was disrupted and uninterpretable. This indicated that at least the F77N and F77Y mutations had not affected the flipping and dU binding of UDG. All UDG variants studied showed a phase of Trp fluorescence recovery that happened at approximately the same rate as the steady state rate of catalysis. These results indicate the active-site phenylalanine has a small, but significant, contribution to transition state stabilization. The large effect of the F77N mutation could possibly be attributed to this polar group perturbing the nonpolar environment of the normal UDG active site. ABSTRACT: These results have implications for proposed UDG reaction mechanisms and demonstrate how seemingly small changes in an enzyme?s active site can affect a finely tuned catalyst. Text (Electronic thesis) in PDF format. System requirements: World Wide Web browser and PDF reader. Mode of access: World Wide Web. Title from title page of source document. Thesis (Ph.D.)--University of Florida, 2003. Includes vita. Includes bibliographical references.
DNA repair proteins conduct a genome-wide search to detect and repair sites of DNA damage wherever they occur. Human alkyladenine DNA glycosylase (AAG) is responsible for recognizing a variety of base lesions, including alkylated and deaminated purines, and initiating their repair via the base excision repair pathway. We have investigated the mechanism by which AAG locates sites of damage using an oligonucleotide substrate containing two sites of DNA damage. This substrate was designed so that AAG randomly binds to either of the two lesions. AAG-catalyzed base excision creates a repair intermediate, and the subsequent partitioning between dissociation and diffusion to the second site can be quantified from the rates of formation of the different products. Our results demonstrate that AAG has the ability to slide for short distances along DNA at physiological salt concentrations. The processivity of AAG decreases with increasing ionic strength to become fully distributive at high ionic strengths, suggesting that electrostatic interactions between the negatively charged DNA and the positively charged DNA binding surface are important for nonspecific DNA binding. Although the amino terminus of the protein is dispensable for glycosylase activity at a single site, we find that deletion of the 80 amino-terminal amino acids significantly decreases the processivity of AAG. These observations support the idea that diffusion on undamaged DNA contributes to the search for sites of DNA damage.
Many enzymes acting on specific rare lesions in DNA are suggested to search for their targets by facilitated one-dimensional diffusion. We have used a recently developed correlated cleavage assay to investigate whether this mechanism operates for Fpg and OGG1, two structurally unrelated DNA glycosylases that excise an important oxidative lesion, 7,8-dihydro-8-oxoguanine (8-oxoG), from DNA. Similar to a number of other DNA glycosylases or restriction endonucleases, Fpg and OGG1 processively excised 8-oxoG from pairs with cytosine at low salt concentrations, indicating that the lesion search likely proceeds by one-dimensional diffusion. At high salt concentrations, both enzymes switched to a distributive mode of lesion search. Correlated cleavage of abasic site-containing substrates proceeded in the same manner as cleavage of 8-oxoG. Interestingly, both Fpg and especially OGG1 demonstrated higher processivity if the substrate contained 8-oxoG.A pairs, against which these enzyme discriminate. Introduction of a nick into the substrate DNA did not decrease the extent of correlated cleavage, suggesting that the search probably involves hopping between adjacent positions on DNA rather than sliding along DNA. This was further supported by the observation that mutant forms of Fpg (Fpg-F110A and Fpg-F110W) with different sizes of the side chain of the amino acid residue inserted into DNA during scanning were both less processive than the wild-type enzyme. In conclusion, processive cleavage by Fpg and OGG1 does not correlate with their substrate specificity and under nearly physiological salt conditions may be replaced with the distributive mode of action.
The first step in the ubiquitous cellular process of nucleotide excision-repair must involve the recognition of a lesion or structural distortion in DNA. This is followed by incision in the strand perceived as damaged; and then coordinated steps of local degradation and re-synthesis occur to replace the defective DNA segment with a new stretch of nucleotides, making use of the intact complementary strand as template. The repair patch is ultimately ligated at its 3' end to the contiguous preexisting DNA strand to restore the integrity of the normal DNA structure. Crucial to this repair scheme is the fact that the genome consists of double-stranded DNA, so that when one strand is damaged the information for its repair can, in principle, be recovered from the other strand. We will review a bit of the early speculation about the nature of the damage recognition step and then discuss the complexity of that event as we currently understand it. An important conceptual contribution to this field resulted from my collaboration with Robert Haynes in which we suggested that "the recognition step in the repair mechanism could be formally equivalent to threading the DNA through a close-fitting 'sleeve' which gauges the closeness-of-fit to the Watson-Crick structure" (Hanawalt and Haynes, 1965).
Escherichia coli uracil-DNA glycosylase was shown to catalyze the hydrolysis of a site-specific uracil residue from a defined single-stranded oligonucleotide (25-mer). With duplex 25-mer, the rate of uracil removal from double-stranded DNA containing a U.G mispair was approximately 2-fold greater than a U.A base pair. The mechanism by which E. coli and rat liver mitochondrial uracil-DNA glycosylase located sequential uracil residues within double-stranded DNA was investigated. Two concatemeric polynucleotide substrates were constructed by ligation of homologous 5'-end 32P-labeled 25-mer double-stranded oligonucleotides that contained either a site-specific U.G or U.A target site at intervals of 25 nucleotides along one strand of the DNA. Reaction of uracil-DNA glycosylase with these concatemeric DNAs, followed by alkaline hydrolysis of the resultant AP-sites, would produce predominantly [32P]25-mer products, if a processive mechanism was used to locate successive uracil residues, or oligomeric multiples of [32P]25-mer, if a distributive mode was exhibited. Both the bacterial and the mitochondrial enzymes were found to act processively on U.A- and U.G-containing DNA in the absence of NaCl, based on the initial rate of 25-mer produced relative to the total amount of uracil excised. Approximately 50% of the total uracil excised resulted in the release of 25-mer product. The addition of NaCl (> or = 50 mM) caused reduced processivity on both U.A- and U.G-containing DNA substrates. The mode of action of uracil-DNA glycosylase was very similar to that observed for the EcoRI endonuclease cleavage of restriction sites contained in the same DNA substrate which was used as a positive control.
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.
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.
Uracil DNA N-glycosylase (UDG) has been used as a model enzyme to test a novel universal approach to discriminate between two possible enzymatic mechanisms of specific site location in DNA, processive (DNA-scanning mechanism) and distributive (random diffusion-mediated mechanism). Two double-stranded concatemeric polynucleotides of defined length (440-480 nucleotides) containing deoxyuridine at either every 10th or 20th nucleotide in the DNA chain were prepared by the ligation of self-complementary 10- or 20-mer oligodeoxyribonucleotides. Incubation of these polynucleotides with Escherichia coli UDG, followed by thermal breakage of the abasic sites, formed fragments that were multiples of either the 10- or the 20-mer. Since the processive and distributive mechanisms of uracil removal by UDG would be very different, the fragment distribution, generated at each time interval during the UDG reaction, should be unique. To show this, we developed a computer model illustrating both possible mechanisms of UDG functioning. The distribution of DNA fragments experimentally generated during the time course of the UDG reaction was compared with the results of the computer programs that modeled the distributive and processive mechanisms. The data indicated that uracil removal, catalyzed by UDG, is consistent with a distributive model.
The bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) protein inactivates uracil-DNA glycosylase (Ung) by forming an exceptionally stable protein-protein complex in which Ugi mimics electronegative and structural features of duplex DNA (Beger, R. D., Balasubramanian, S., Bennett, S. E., Mosbaugh, D. W., and Bolton, P. H. (1995) J. Biol. Chem. 270, 16840-16847; Mol, C. D., Arvai, A. S., Sanderson, R. J., Slupphaug, G., Kavli, B., Krokan, H. E., Mosbaugh, D. W., and Tainer, J. A. (1995) Cell 82, 701-708). The role of specific carboxylic amino acid residues in forming the Ung.Ugi complex was investigated using selective chemical modification techniques. Ugi treated with carbodiimide and glycine ethyl ester produced five discrete protein species (forms I-V) that were purified and characterized. Analysis by mass spectrometry revealed that Ugi form I escaped protein modification, and forms II-V showed increasing incremental amounts of acyl-glycine ethyl ester adduction. Ugi forms II-V retained their ability to form a Ung.Ugi complex but exhibited a reduced ability to inactivate Escherichia coli Ung, directly reflecting the extent of modification. Competition experiments using modified forms II-V with unmodified Ugi as a competitor protein revealed that unmodified Ugi preferentially formed complex. Furthermore, unmodified Ugi and poly(U) were capable of displacing forms II-V from a preformed Ung.Ugi complex but were unable to displace Ugi form I. The primary sites of acyl-glycine ethyl ester adduction were located in the alpha2-helix of Ugi at Glu-28 and Glu-31. We infer that these two negatively charged amino acids play an important role in mediating a conformational change in Ugi that precipitates the essentially irreversible Ung/Ugi interaction.
A DNA endonuclease, isolated from the nuclei of normal human and xeroderma pigmentosum complementation group A (XPA) cells, which recognizes predominately pyrimidine dimers, was examined for the mechanism by which it locates sites of damage on UVC-irradiated DNA. In reaction mixtures with low ionic strengths (i.e. lacking KCl), the normal and XPA endonuclease locate sites of UV damage on both naked and reconstituted nucleosomal DNA by different mechanisms. On both of these substrates, the normal endonuclease acts by a processive mechanism, meaning that it binds non-specifically to DNA and scans the DNA for sites of damage, whereas the XPA endonuclease acts by a distributive one, meaning that it randomly locates sites of damage on DNA. However, while both the normal and XPA endonucleases can incise UVC irradiated naked DNA, they differ in ability to incise damaged nucleosomal DNA. The normal endonuclease showed increased activity on UVC treated nucleosomal DNA compared with naked DNA, whereas the XPA endonuclease showed decreased activity on the damaged nucleosomal substrate. Since a processive mechanism of action is sensitive to the ionic strength of the micro-environment, the KCl concentration of the reaction was increased. At 70 mM KCI, the normal endonuclease switched to a distributive mechanism of action and its ability to incise damaged nucleosomal DNA also decreased. These studies show that there is a correlation between the ability of these endonucleases to act by a processive mechanism and their ability to incise damaged nucleosomal DNA; the normal endonuclease, which acts processively, can incise damaged nucleosomal DNA, whereas the XPA endonuclease, which acts distributively, is defective in ability to incise this substrate.
A wide range of cytotoxic and mutagenic DNA bases are removed by different DNA glycosylases, which initiate the base excision repair pathway. DNA glycosylases cleave the N-glycosylic bond between the target base and deoxyribose, thus releasing a free base and leaving an apurinic/apyrimidinic (AP) site. In addition, several DNA glycosylases are bifunctional, since they also display a lyase activity that cleaves the phosphodiester backbone 3' to the AP site generated by the glycosylase activity. Structural data and sequence comparisons have identified common features among many of the DNA glycosylases. Their active sites have a structure that can only bind extrahelical target bases, as observed in the crystal structure of human uracil-DNA glycosylase in a complex with double-stranded DNA. Nucleotide flipping is apparently actively facilitated by the enzyme. With bacteriophage T4 endonuclease V, a pyrimidine-dimer glycosylase, the enzyme gains access to the target base by flipping out an adenine opposite to the dimer. A conserved helix-hairpin-helix motif and an invariant Asp residue are found in the active sites of more than 20 monofunctional and bifunctional DNA glycosylases. In bifunctional DNA glycosylases, the conserved Asp is thought to deprotonate a conserved Lys, forming an amine nucleophile. The nucleophile forms a covalent intermediate (Schiff base) with the deoxyribose anomeric carbon and expels the base. Deoxyribose subsequently undergoes several transformations, resulting in strand cleavage and regeneration of the free enzyme. The catalytic mechanism of monofunctional glycosylases does not involve covalent intermediates. Instead the conserved Asp residue may activate a water molecule which acts as the attacking nucleophile.
Rotation of a DNA nucleotide out of the double helix and into a protein binding pocket ("base flipping") was first observed in the structure of a DNA methyltransferase. There is now evidence that a variety of proteins, particularly DNA repair enzymes, use base flipping in their interactions with DNA. Though the mechanisms for base movement into extrahelical positions are still unclear, the focus of this review is how base recognition is modulated by the stringency of binding to the extrahelical base(s) or sugar moiety.
Exposure to UVA radiation of SV40 DNA substituted with bromodeoxyuridine (BrdU) in the presence of Hoechist dye 33258 results
in the production of uracil. The yield of uracil was determined by measuring the increase in the single-strand break (SSB)
yield after incubation of the photolyzed DNA with uracil-DNA glycosylase (UDG) in the presence of the tripeptide lysyl-tyrosyl-lysine
(KYK). UDG removes uracil to leave an abasic site which is then cleaved to a SSB by KYK. The SSB yield was quantified by digital
video imaging of ethidium fluorescence after separation of the I, II and III forms of SV40 DNA by agarose gel electrophoresis.
Uracil is not detected when photolysis is carried out in the absence of the dye nor when unsubstituted DNA is used as the
substrate. Without UDG or KYK treatment, the F0 for the loss of form I DNA is 100 J/m2. This falls to 13 J/m2 after incubation with UDG and KYK, indicating that uracil formation is ˜5-fold more efficient than SSB formation. Formation
of uracil suggests a mechanism for the high cellular toxicity of the dye-BrdU-UVA treatment.
Site-directed mutants of the herpes simplex virus type 1 uracil-DNA glycosylase lacking catalytic activity have been used to probe the substrate recognition of this highly conserved and ubiquitous class of DNA-repair enzyme utilizing surface plasmon resonance. The residues aspartic acid-88 and histidine-210, implicated in the catalytic mechanism of the enzyme (Savva, R., McAuley-Hecht, K., Brown, T., and Pearl, L. (1995) Nature 373, 487-493; Slupphaug, G., Mol, C. D., Kavli, B., Arvai, A. S., Krokan, H. E. and Tainer, J. A. (1996) Nature 384, 87-92) were separately mutated to asparagine to allow investigations of substrate recognition in the absence of catalysis. The mutants were shown to be correctly folded and to lack catalytic activity. Binding to single- and double-stranded oligonucleotides, with or without uracil, was monitored by real-time biomolecular interaction analysis using surface plasmon resonance. Both mutants exhibited comparable rates of binding and dissociation on the same uracil-containing substrates. Interaction with single-stranded uracil-DNA was found to be stronger than with double-stranded uracil-DNA, and the binding to Gua:Ura mismatches was significantly stronger than that to Ade:Ura base pairs suggesting that the stability of the base pair determines the efficiency of interaction. Also, there was negligible interaction between the mutants and single- or double-stranded DNA lacking uracil, or with DNA containing abasic sites. These results suggest that it is uracil in the DNA, rather than DNA itself, that is recognized by the uracil-DNA glycosylases.
The DNA repair enzyme uracil DNA glycosylase (UDG) catalyzes hydrolytic cleavage of the N-glycosidic bond of premutagenic uracil residues in DNA by flipping the uracil base from the DNA helix. The mechanism of base flipping and the role this step plays in site-specific DNA binding and catalysis by enzymes are largely unknown. The thermodynamics and kinetics of DNA binding and uracil flipping by UDG have been studied in the absence of glycosidic bond cleavage using substrate analogues containing the 2'-alpha and 2'-beta fluorine isomers of 2'-fluoro-2'-deoxyuridine (Ubeta, Ualpha) positioned adjacent to a fluorescent nucleotide reporter group 2-aminopurine (2-AP). Activity measurements show that DNA containing a Ubeta or Ualpha nucleotide is a 10(7)-fold slower substrate for UDG (t1/2 approximately 20 h), which allows measurements of DNA binding and base flipping in the absence of glycosidic bond cleavage. When UDG binds these analogues, but not other DNA molecules, a 4-8-fold 2-AP fluorescence enhancement is observed, as expected for a decrease in 2-AP base stacking resulting from enzymatic flipping of the adjacent uracil. Thermodynamic measurements show that UDG forms weak nonspecific complexes with dsDNA (KDns = 1.5 microM) and binds approximately 25-fold more tightly to Ubeta containing dsDNA (KDapp approximately 50 nM). Thus, base flipping contributes less than approximately 2 kcal/mol to the free energy of binding and is not a major component of the >10(6)-fold catalytic specificity of UDG. Kinetic studies at 25 degrees C show that site-specific binding occurs by a two-step mechanism. The first step (E + S left and right arrow ES) involves the diffusion-controlled binding of UDG to form a weak nonspecific complex with the DNA (KD approximately 1.5-3 microM). The second step (ES left and right arrow E'F) involves a rapid step leading to reversible uracil flipping (kmax approximately 1200 s-1). This step is followed closely by a conformational change in UDG that was monitored by the quenching of tryptophan fluorescence. The results provide evidence for an enzyme-assisted mechanism for uracil flipping and exclude a passive mechanism in which the enzyme traps a transient extrahelical base in the free substrate. The data suggest that the duplex structure of the DNA is locally destabilized before the base-flipping step, thereby facilitating extrusion of the uracil. Thus, base flipping contributes little to the free energy of DNA binding but contributes greatly to specificity through an induced-fit mechanism.
One of the major DNA repair pathways is base excision repair, in which DNA bases that have been damaged by endogenous or exogenous agents are removed by the action of a class of enzymes known as DNA glycosylases. One subset of the known DNA glycosylases has an associated abasic lyase activity that generates a phosphodiester bond scission. The base excision pathway is completed by the sequential action of abasic endonucleases, DNA polymerases, and DNA ligases. Base excision repair of ultraviolet (UV) light-induced dipyrimidine photoproducts has been described in a variety of prokaryotic and eukaryotic organisms and phages. These enzymes vary significantly in their exact substrate specificity and in the catalytic mechanism by which repair is initiated. The prototype enzyme within this class of UV-specific DNA glycosylases is T4 endonuclease V. Endonuclease V holds the distinction of being the first glycosylase (1) to have its structure solved by X-ray diffraction of the enzyme alone as well as in complex with pyrimidine dimer-containing DNA, (2) to have its key catalytic active site residues identified, and (3) to have its mechanism of target DNA site location determined and the biological relevance of this process established. Thus, the study of endonuclease V has been critical in gaining a better understanding of the mechanisms of all DNA glycosylases.
The role of the conserved histidine-187 located in the leucine intercalation loop of Escherichia coli uracil-DNA glycosylase (Ung) was investigated. Using site-directed mutagenesis, an Ung H187D mutant protein was created, overproduced, purified to apparent homogeneity, and characterized in comparison to wild-type Ung. The properties of Ung H187D differed from Ung with respect to specific activity, substrate specificity, DNA binding, pH optimum, and inhibition by uracil analogues. Ung H187D exhibited a 55000-fold lower specific activity and a shift in pH optimum from pH 8.0 to 7.0. Under reaction conditions optimal for wild-type Ung (pH 8.0), the substrate preference of Ung H187D on defined single- and double-stranded oligonucleotides (25-mers) containing a site-specific uracil target was U/G-25-mer > U-25-mer > U/A-25-mer. However, Ung H187D processed these same DNA substrates at comparable rates at pH 7.0 and the activity was stimulated approximately 3-fold relative to the U-25-mer substrate. Ung H187D was less susceptible than Ung to inhibition by uracil, 6-amino uracil, and 5-fluorouracil. Using UV-catalyzed protein/DNA cross-linking to measure DNA binding affinity, the efficiency of Ung H187D binding to thymine-, uracil-, and apyrimidinic-site-containing DNA was (dT20) = (dT19-U) >/= (dT19-AP). Comparative analysis of the biochemical properties and the X-ray crystallographic structures of Ung and Ung H187D [Putnam, C. D., Shroyer, M. J. N., Lundquist, A. J., Mol, C. D., Arvai, A. S., Mosbaugh, D. W., and Tainer, J. A. (1999) J. Mol. Biol. 287, 331-346] provided insight regarding the role of His-187 in the catalytic mechanism of glycosylic bond cleavage. A novel mechanism is proposed wherein the developing negative charge on the uracil ring and concomitant polarization of the N1-C1' bond is sustained by resonance effects and hydrogen bonding involving the imidazole side chain of His-187.
Interaction of DNA repair proteins with damaged DNA in eukaryotic cells is influenced by the packaging of DNA into chromatin. The basic repeating unit of chromatin, the nucleosome, plays an important role in regulating accessibility of repair proteins to sites of damage in DNA. There are a number of different pathways fundamental to the DNA repair process. Elucidation of the proteins involved in these pathways and the mechanisms they utilize for interacting with damaged nucleosomal and nonnucleosomal DNA has been aided by studies of genetic diseases where there are defects in the DNA repair process. Two of these diseases are xeroderma pigmentosum (XP) and Fanconi anemia (FA). Cells from patients with these disorders are similar in that they have defects in the initial steps of the repair process. However, there are a number of important differences in the nature of these defects. One of these is in the ability of repair proteins from XP and FA cells to interact with damaged nucleosomal DNA. In XP complementation group A (XPA) cells, for example, endonucleases present in a chromatin-associated protein complex involved in the initial steps in the repair process are defective in their ability to incise damaged nucleosomal DNA, but, like the normal complexes, can incise damaged naked DNA. In contrast, in FA complementation group A (FA-A) cells, these complexes are equally deficient in their ability to incise damaged naked and similarly damaged nucleosomal DNA. This ability to interact with damaged nucleosomal DNA correlates with the mechanism of action these endonucleases use for locating sites of damage. Whereas the FA-A and normal endonucleases act by a processive mechanism of action, the XPA endonucleases locate sites of damage distributively. Thus the mechanism of action utilized by a DNA repair enzyme may be of critical importance in its ability to interact with damaged nucleosomal DNA.
Apurinic/apyrimidinic endonuclease (AP endo) is believed to play a critical role in repair of oxidative damage of DNA and is proposed to initiate repair of most abasic sites in the base excision repair pathway. AP endo makes a single nick 5' to an abasic site in double-stranded DNA. In this study, we investigated whether AP endo locates an abasic site through a processive or a distributive mechanism. We used a linear multi-abasic site substrate (concatemer), synthesized by ligating together identical 25-nucleotide monomeric units (25-mers). We first determined that the 25-mer monomer from which the concatemers were prepared was nicked by AP endo in a fashion similar to that of the previously published 49-mer substrate with a different sequence. Steady state parameters K(m) and k(cat) and single-turnover parameters for substrate binding were comparable to previously published values. Using the multi-abasic site concatemer, we demonstrated that AP endo was capable of cleaving approximately seven to eight abasic sites, traveling at least 200 nucleotides, before dissociating from its substrate. Thus, AP endo, like uracil DNA glycosylase, behaves in a quasi processive fashion. Processivity could be separated from catalysis, since processivity was maximal at 25 mM NaCl, while the rate of cleavage was maximal at 125 mM salt. In short, nicking activity was maximized close to physiological salt molarities while processivity was midrange at physiological salt concentrations. The latter is likely to be subject to tight regulation by small changes in ionic strength.
We have previously shown that endonucleases present in a protein complex, which has specificity for cyclobutane pyrimidine dimers, locate sites of damage in DNA by a processive mechanism of action in normal human lymphoblastoid cells. In contrast, the endonucleases present in this complex from xeroderma pigmentosum complementation group A (XPA) cells locate damage sites by a distributive or significantly less processive mechanism. Since the XPA protein has been shown to be responsible for the DNA repair defect in XPA cells, this protein was examined for involvement in the mechanism of target site location of these endonucleases. A recombinant XPA protein, produced by expression of the normal XPA cDNA in E. coli, was isolated and purified. The results show that the recombinant XPA protein was able to correct the defect in ability of the XPA endonucleases to act by a processive mechanism of action on UVC irradiated DNA. These studies indicate that the XPA protein, in addition to a role in damage recognition or damage verification, may function as a processivity factor.
Human alkyladenine glycosylase (AAG) and Escherichia coli 3-methyladenine glycosylase (AlkA) are base excision repair glycosylases that recognize and excise a variety of alkylated bases from DNA. The crystal structures of these enzymes have provided insight into their substrate specificity and mechanisms of catalysis. Both enzymes utilize DNA bending and base-flipping mechanisms to expose and bind substrate bases. Crystal structures of AAG complexed to DNA suggest that the enzyme selects substrate bases through a combination of hydrogen bonding and the steric constraints of the active site, and that the enzyme activates a water molecule for an in-line backside attack of the N-glycosylic bond. In contrast to AAG, the structure of the AlkA-DNA complex suggests that AlkA substrate recognition and catalytic specificity are intimately integrated in a S(N)1 type mechanism in which the catalytic Asp238 directly promotes the release of modified bases.
Escherichia coli double-strand uracil-DNA glycosylase (Dug) was purified to apparent homogeneity as both a native and recombinant protein. The molecular weight of recombinant Dug was 18 670, as determined by matrix-assisted laser desorption-ionization mass spectrometry. Dug was active on duplex oligonucleotides (34-mers) that contained site-specific U.G, U.A, ethenoC.G, and ethenoC.A targets; however, activity was not detected on DNA containing a T.G mispair or single-stranded DNA containing either a site-specific uracil or ethenoC residue. One of the distinctive characteristics of Dug was that the purified enzyme excised a near stoichiometric amount of uracil from U.G-containing oligonucleotide substrate. Electrophoretic mobility shift assays revealed that the lack of turnover was the result of strong binding by Dug to the reaction product apyrimidinic-site (AP) DNA. Addition of E. coli endonuclease IV stimulated Dug activity by enhancing the rate and extent of uracil excision by promoting dissociation of Dug from the AP. G-containing 34-mer. Catalytically active endonuclease IV was apparently required to mediate Dug turnover, since the addition of 5 mM EDTA mitigated the effect. Further support for this interpretation came from the observations that Dug preferentially bound 34-mer containing an AP.G target, while binding was not observed on a substrate incised 5' to the AP-site. We also investigated whether Dug could initiate a uracil-mediated base excision repair pathway in E. coli NR8052 cell extracts using M13mp2op14 DNA (form I) containing a site-specific U.G mispair. Analysis of reaction products revealed a time dependent appearance of repaired form I DNA; addition of purified Dug to the cell extract stimulated the rate of repair.
Base flipping is a highly conserved process by which enzymes swivel an entire nucleotide from the DNA base stack into their
active site pockets. Uracil DNA glycosylase (UDG) is a paradigm enzyme that uses a base flipping mechanism to catalyze the
hydrolysis of the N-glycosidic bond of 2′-deoxyuridine (2′-dUrd) in DNA as the first step in uracil base excision repair. Flipping of 2′-dUrd
by UDG has been proposed to follow a “pushing” mechanism in which a completely conserved leucine side chain (Leu-191) is inserted
into the DNA minor groove to expel the uracil. Here we report a novel implementation of the “chemical rescue” approach to
show that the weak binding affinity and low catalytic activity of L191A or L191G can be completely or partially restored by
substitution of a pyrene (Y) nucleotide wedge on the DNA strand opposite to the uracil base (U/A to U/Y). These results indicate
that pyrene acts both as a wedge to push the uracil from the base stack in the free DNA and as a “plug” to hinder its reinsertion
after base flipping. Pyrene rescue should serve as a useful and novel tool to diagnose the functional roles of other amino
acid side chains involved in base flipping.
The action of the dimer-specific endonuclease V of bacteriophage T4 was studied on UV-irradiated, covalently-closed circular
DNA. Form I ColEl DNA preparations containing average dimer frequencies ranging from 2.5 to 35 pyrimidine dimers per molecule
were treated with T4 endonuclease V and analysed by agarose gel electrophoresis. At all dimer frequencies examined, the production
of form III DNA was linear with time and the double-strand scissions were made randomly on the ColEl DNA genome. Since the
observed fraction of form III DNA increased with increasing dimer frequency but the initial rate of loss of form I decreased
with increasing dimer frequency, it was postulated that multiple single-strand scissions could be produced in a subset of
the DNA population while some DNA molecules contained no scissions. When DNA containing an average of 25 dimers per circle
was incubated with limiting enzyme concentrations, scissions appeared at most if not all dimer sites in some molecules before
additional strand scissions were produced in other DNA molecules. The results support a processive model for the interaction
of T4 endonuclease V with UV-irradiated DNA.
Uracil-DNA glycosidase, an enzyme that catalyzes the release of free uracil from uracil-containing DNA, has been purified 11,000-fold from E. coli cell extracts. The enzyme preparation was essentially homogenous, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native enzyme is a small monomeric protein of a molecular weight of 24,500 ± 1,000. The enzyme efficiently releases uracil from single-stranded DNA and also from double-stranded DNA with uracil residues hydrogen-bonded to either adenine or guanine residues. On the other hand, DNA molecules containing 5-bromouracil residues, pyrimidine dimers, or deaminated purine residues are not substrates. Uracil-DNA glycosidase does not cleave free dUMP at a detectable rate and shows little activity with uracil-containing oligonucleotides. It has no cofactor dependence and apparently acts by hydrolytic cleavage of the base-sugar bonds in dUMP residues, as there is no incorporation of phosphate or pyrimidines in the DNA when uracil is released. When the enzyme was incubated with covalently closed circular DNA containing a small number of uracil residues introduced by deamination of cytosine residues with bisulfite, alkali-labile sites, but no chain breaks were introduced in the DNA molecules. Such DNA molecules could subsequently be cleaved by an endonuclease that specifically attacks DNA at apurinic and apyrimidinic sites, E. coli endonuclease IV. These data indicate that uracil-DNA glycosidase functions in the repair of DNA containing accidentally introduced uracil residues.
Several DNA-interactive proteins, including the DNA repair enzyme T4 endonuclease V, have been shown to locate their target recognition sites utilizing an electrostatically mediated facilitated diffusion mechanism. Previous work indicates that a decrease in the affinity of endonuclease V for nontarget DNA results in an increased nontarget dissociation rate. This study was designed to investigate the effect of an increase in the affinity of endonuclease V for nontarget DNA. Using a working structural model of the enzyme as a guide, the electrostatic character of endonuclease V was altered. Substitution of Thr-7 with Lys-7 resulted in an enzyme with wild type in vitro characteristics. Mutations which increased the positive charge along a proposed solvent-exposed alpha-helical face had significant effects. The mutants Ala-30, Val-31----Lys-30, Leu-31 and Asn-37----Lys-37 displayed wild type in vitro apurinic-specific and dimer-specific nicking activities. Although the processive dimer-specific nicking rate of the Lys-37 mutant resembled that of wild type, the rate of the Lys-30, Leu-31 mutant was reduced by 60%. In addition, the salt concentration range over which these mutants processively nick dimer-containing DNA has been greatly expanded. Both mutants are shown to have an increased affinity for nontarget DNA.
T4 endonuclease V is a pyrimidine dimer-specific DNA repair enzyme which has been previously shown not to require metal ions for either of its two catalytic activities or its DNA binding function by virtue of its ability to function in the presence of metal-chelating agents. However, we have investigated whether the single cysteine within the enzyme was able to bind metal salts and influence the various activities of this repair enzyme. A series of metals (Hg2+, Ag+, Cu+) were shown to inactivate both endonuclease Vs pyrimidine dimer-specific DNA glycosylase activity and the subsequent apurinic nicking activity. The binding of metal to endonuclease V did not interfere with nontarget DNA scanning or pyrimidine dimer-specific binding. The Cys-78 codon within the endonuclease V gene was changed by oligonucleotide site-directed mutagenesis to Thr-78 and Ser-78 in order to determine whether the native cysteine was directly involved in the enzyme's DNA catalytic activities and whether the cysteine was primarily responsible for the metal binding. The mutant enzymes were able to confer enhanced ultraviolet light (UV) resistance to DNA repair-deficient Escherichia coli at levels equal to that conferred by the wild type enzyme. The C78T mutant enzyme was purified to homogeneity and shown to be catalytically active on pyrimidine dimer-containing DNA. The catalytic activities of the C78T mutant enzyme were demonstrated to be unaffected by the addition of Hg2+ or Ag+ at concentrations 1000-fold greater than that required to inhibit the wild type enzyme. These data suggest that the cysteine is not required for enzyme activity but that the binding of certain metals to that amino acid block DNA incision by either preventing a conformational change in the enzyme after it has bound to a pyrimidine dimer or sterically interfering with the active site residue's accessibility to the pyrimidine dimer.
Facilitated diffusion along nontarget DNA is employed by numerous DNA-interactive proteins to locate specific targets. Until now, the biological significance of DNA scanning has remained elusive. T4 endonuclease V is a DNA repair enzyme which scans nontarget DNA and processively incises DNA at the site of pyrimidine dimers which are produced by exposure to ultraviolet (UV) light. In this study we tested the hypothesis that there exists a direct correlation between the degree of processivity of wild type and mutant endonuclease V molecules and the degree of enhanced UV resistance which is conferred to repair-deficient Eshcerichia coli. This was accomplished by first creating a series of endonuclease V mutants whose in vitro catalytic activities were shown to be very similar to that of the wild type enzyme. However, when the mechanisms by which these enzymes search nontarget DNA for its substrate were analyzed in vitro and in vivo, the mutants displayed varying degrees of nontarget DNA scanning ranging from being nearly as processive as wild type to randomly incising dimers within the DNA population. The ability of these altered endonuclease V molecules to enhance UV survival in DNA repair-deficient E. coli then was assessed. The degree of enhanced UV survival was directly correlated with the level of facilitated diffusion. This is the first conclusive evidence directly relating a reduction of in vivo facilitated diffusion with a change in an observed phenotype. These results support the assertion that the mechanisms which DNA-interactive proteins employ in locating their target sites are of biological significance.
Micrococcus luteus UV endonuclease incises DNA at the sites of ultraviolet (UV) light-induced pyrimidine dimers. The mechanism of incision has been previously shown to be a glycosylic bond cleavage at the 5'-pyrimidine of the dimer followed by an apyrimidine endonuclease activity which cleaves the phosphodiester backbone between the pyrimidines. The process by which M. luteus UV endonuclease locates pyrimidine dimers within a population of UV-irradiated plasmids was shown to occur, in vitro, by a processive or "sliding" mechanism on non-target DNA as opposed to a distributive or "random hit" mechanism. Form I plasmid DNA containing 25 dimers per molecule was incubated with M. luteus UV endonuclease in time course reactions. The three topological forms of plasmid DNA generated were analyzed by agarose gel electrophoresis. When the enzyme encounters a pyrimidine dimer, it is significantly more likely to make only the glycosylase cleavage as opposed to making both the glycosylic and phosphodiester bond cleavages. Thus, plasmids are accumulated with many alkaline-labile sites relative to single-stranded breaks. In addition, reactions were performed at both pH 8.0 and pH 6.0, in the absence of NaCl, as well as 25,100, and 250 mM NaCl. The efficiency of the DNA scanning reaction was shown to be dependent on both the ionic strength and pH of the reaction. At low ionic strengths, the reaction was shown to proceed by a processive mechanism and shifted to a distributive mechanism as the ionic strength of the reaction increased. Processivity at pH 8.0 is shown to be more sensitive to increases in ionic strength than reactions performed at pH 6.0.
In 1970 Riggs et al. (1) reported that Escherichia coli lac repressor binding to λ DNA in vitro seemed to find its target (operator) site on the DNA at a rate as much as 1000-fold faster than the upper limit estimated for a diffusion-controlled process involving macromolecules of this size. This observation startled and intrigued many physically oriented molecular biologists and biochemists and initiated a flurry of theoretical and experimental papers seeking to offer an explanation. However, scrutiny of the older literature reveals that scientists, ranging from mathematicians to biologists, had long been concerned with how systems of various sorts might transcend the rate limits set by three-dimensional diffusion control (2).
Such problems are now of interest at many different levels. The pure physical chemist feels that an understanding of such phenomena might provide new insight into what happens when molecules meet and rearrange in the course of forming and passing through the transition state complex. The enzyme mechanician hopes that the secrets of some of the astonishing increases in rates achieved in enzyme-catalyzed reactions may be revealed by a study of these rate accelerations. And the cell biologist who studies macromolecular interactions and assembly processes is intrigued by the possibility that these systems may reveal opportunities for acceleration of intracellular rates beyond the limits set by the relatively slow diffusion of macromolecules in the cytoplasm.
In this minireview we propose to touch on recent progress in all of these areas but will focus primarily on a problem that has engaged our attention over the past few years, i.e. how do protein regulators of gene expression at the transcriptional level find their regulatory DNA targets at speeds that appear to be faster than diffusion controlled?
The potential for processive EcoRI endonuclease hydrolysis has been examined on several DNA substrates containing two EcoRI sites which were embedded in identical sequence environments. With a 388-base pair circular DNA, in which the two recognition sites are separated by 51 base pairs (shorter distance) or 337 base pairs (longer distance), 77 and 34% of all events involved processive hydrolysis at ionic strengths of 0.059 and 0.13, respectively. However, the frequency of processive action on linear substrates, in which the two sites were separated by 51 base pairs, was only 42 and 17% at these ionic strengths, values half those observed with the circular DNA. Processive action was not detectable on circular or linear substrates at an ionic strength of 0.23. These findings indicate that DNA search by the endonuclease occurs by facilitated diffusion, a mechanism in which the protein locates and leaves its recognition sequence by interacting with nonspecific DNA sites. We suggest that processivity on linear substrates is limited to values half that for small circles due to partitioning of the enzyme between the two products generated by cleavage of a linear molecule. Given such topological effects, measured processivity values imply that the endonuclease can diffuse within a DNA domain to locate and recognize an EcoRI site 50 to 300 base pairs distant from an initial binding site, with minimum search efficiencies being 80 and 30% at ionic strengths of 0.059 and 0.13, respectively. The high efficiency of processive action indicates that a positionally correlated mode of search plays a major role in facilitated diffusion in this system under such conditions. Also consistent with this view was the identification of a striking position effect when two closely spaced EcoRI sites were asymmetrically positioned near the end of a linear DNA. The endonuclease displays a substantial preference for the more centrally located recognition sequence. This preference does not reflect differential sensitivity of the two sites to cleavage per se, but can be simply explained by preferential entry of the enzyme via the larger nonspecific target available to the more centrally positioned recognition sequence. These conclusions differ from those of a previous qualitative analysis of endonuclease processivity over short distances (Langowski, J., Alves, J., Pingoud, A., and Maass, G. (1983) Nucleic Acids Res. 11, 501-513).
In this study, a novel approach to the analysis of DNA repair in Escherichia coli was employed which allowed the first direct determination of the mechanisms by which endogenous DNA repair enzymes encounter target sites in vivo. An in vivo plasmid DNA repair analysis was employed to discriminate between two possible mechanisms of target site location: a processive DNA scanning mechanism or a distributive random diffusion mechanism. The results demonstrate that photolyase acts by a distributive mechanism within E. coli. In contrast, UvrABC-initiated excision repair occurs by a limited processive DNA scanning mechanism. A majority of the dimer sites on a given plasmid molecule were repaired prior to the dissociation of the UvrABC complex. Furthermore, plasmid DNA repair catalyzed by the UvrABC complex occurs without a detectable accumulation of nicked plasmid intermediates despite the fact that the UvrABC complex generates dual incisions in the DNA at the site of a pyrimidine dimer. Therefore, the binding or assembly of the UvrABC complex on DNA at the site of a pyrimidine dimer represents the rate-limiting step in the overall process of UvrABC-initiated excision repair in vivo.
The process by which DNA-interactive proteins locate specific sequences or target sites on cellular DNA within Escherichia coli is a poorly understood phenomenon. In this study, we present the first direct in vivo analysis of the interaction of a DNA repair enzyme, T4 endonuclease V, and its substrate, pyrimidine dimer-containing plasmid DNA, within UV-irradiated E. coli. A pyrimidine dimer represents a small target site within large domains of DNA. There are two possible paradigms by which endonuclease V could locate these small target sites: a processive mechanism in which the enzyme "scans" DNA for dimer sites or a distributive process in which dimers are located by random three-dimensional diffusion. In order to discriminate between these two possibilities in E. coli, an in vivo DNA repair assay was developed to study the kinetics of plasmid DNA repair and the dimer frequency (i.e. the number of dimer sites on a given plasmid molecule) in plasmid DNA as a function of time during repair. Our results demonstrate that the overall process of plasmid DNA repair initiated by T4 endonuclease V (expressed from a recombinant plasmid within repair-deficient E. coli) occurs by a processive mechanism. Furthermore, by reducing the temperature of the repair incubation, the endonuclease V-catalyzed incision step has been effectively decoupled from the subsequent steps including repair patch synthesis, ligation, and supercoiling. By this manipulation, it was determined that the overall processive mechanism is composed of two phases: a rapid processive endonuclease V-catalyzed incision reaction, followed by a slower processive mechanism, the ultimate product of which is the dimer-free supercoiled plasmid molecule.
T4 endonuclease V is a pyrimidine dimer-specific endonuclease which generates incisions in DNA at the sites of pyrimidine dimers by a processive reaction mechanism. A model is presented in which the degree of processivity is directly related to the efficacy of the one-dimensional diffusion of endonuclease V on DNA by which the enzyme locates pyrimidine dimers. The modulation of the processive nicking activity of T4 endonuclease V on superhelical covalently closed circular DNA (form I) which contains pyrimidine dimers has been investigated as a function of the ionic strength of the reaction. Agarose gel electrophoresis was used to separate the three topological forms of the DNA which were generated in time course reactions of endonuclease V with dimer-containing form I DNA in the absence of NaCl, and in 25, 50, and 100 mM NaCl. The degree of processivity was evaluated in terms of the mass fraction of form III (linear) DNA which was produced as a function of the fraction of form I DNA remaining. Processivity is maximal in the absence of NaCl and decreases as the NaCl concentration is increased. At 100 mM NaCl, processivity is abolished and endonuclease V generates incisions in DNA at the site of dimers by a distributive reaction mechanism. The change from the distributive to a processive reaction mechanism occurs at NaCl concentrations slightly below 50 mM. The high degree of processivity which is observed in the absence of NaCl is reversible to the distributive mechanism, as demonstrated by experiments in which the NaCl concentration was increased during the time course reaction. In addition, unirradiated DNA inhibited the incision of irradiated DNA only at NaCl concentrations at which processivity was observed.
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.
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.
We have applied the formalism developed previously for the kinetics of domain-localized reaction [S. Mazur and M. T. Record, Jr. (1986) Biopolymers25, 985–1008] to describe complex mechanisms of association of a protein with a specific site on a large DNA molecule also containing many nonspecific binding sites. These nonspecific sites participate in the mechanism of formation of the specific complex through competitive binding and the facilitating mechanisms of sliding and transfer. The effects of localizing the sites in a domain are represented by a simple algebraic expression, and the sequence of interactions within the domain are described by equations closely related to a conventional, homogeneous solution mechanism. We apply this formalism to examine the interplay between sliding and direct transfer in domain-localized interactions in general and in the lac repressor-lac operator interaction in particular. Experimental investigation of the effect of the molecular location of the specific site (e.g., end vs middle of the polymer chain) on the kinetics of association may allow the contributions of sliding and direct transfer to be resolved.
Brief exposure of covalently closed circular duplex PM2 DNA to low concentrations of the clinical bleomycin mixture (Blenoxane) resulted in specific fragmentation of the genome that does not depend on the presence of superhelical turns. The double-strand breaks are in fact produced at several discrete sites on the PM2 genome but frequently occurring near the HpaII restriction endonuclease cleavage site. Initial rates of formation of nicked circular and linear duplex PM2 DNAs are reduced to different extents as the ionic strength of the reaction is increased. Increasing ionic strength is most effective in reducing the initial rate and overall yield of apparent double-strand scissions compared with single-strand scissions in the bleomycin-treated PM2 DNA.
Mutagenesis has remained an intriguing aspect of genetics since the beginning of this century, and its analysis has proceeded hand in hand with the elucidation of gene replication and expression. Interest in this area has further heightened with the growing awareness that numerous environmental agents may cause mutations in humans. These mutations may lead to metabolic as well as neoplastic diseases. Advances during the past 15 years have revealed two major classes of mutagenic mechanisms: directly induced base mispairing, and misrepair. Alkylating agents for instance, generate many different reaction products in DNA, but only two of these (O6-alkylguanine and O4-alkylthymine) are likely candidates for directly induced mispairing. He has also turned out to be an important mutagen, one that presents a particular serious challenge to large genomes; it converts cytosine to uracil and guanine to an analogue of cytosine. DNA lesions that interrupt DNA chain elongation, including many of other products of alkylation, often trigger an error-prone postreplication repair process. Current evidence suggests that this process involves in incorrect insertion of bases into gaps in progeny-strand DNA opposite such a lesion. Mutagenic mechanisms are subject to powerful genetic controls that include the activities of DNA polymerases in the selection of deoxynucleoside triphosphates and the removal of incorrectly inserted nucleotides.
Facilitated one-dimensional diffusion is a general mechanism utilized by several DNA-interactive proteins as they search for their target sites within large domains of nontarget DNA. T4 endonuclease V is a protein which scans DNA in a nonspecifically bound state and processively incises DNA at ultraviolet (UV)-induced pyrimidine dimer sites. An electrostatic contribution to this mechanism of target location has been established. Previous studies indicate that a decrease in the affinity of endonuclease V for nontarget DNA results in a decreased ability to scan DNA and a concomitant decrease in the ability to enhance UV survival in repair-deficient Escherichia coli. This study was designed to question the contrasting effect of an increase in the affinity of endonuclease V for nontarget DNA. With this as a goal, a gradient of increasingly basic amino acid content was created along a proposed endonuclease V-nontarget DNA interface. This incremental increase in positive charge correlated with the stepwise enhancement of nontarget DNA binding, yet inversely correlated with enhanced UV survival in repair-deficient E. coli. Further analysis suggests that the observed reduction in UV survival is consistent with the hypothesis that enhanced nontarget DNA affinity results in reduced pyrimidine dimer-specific recognition and/or binding. The net effect is a reduction in the efficiency of pyrimidine dimer incision.
In order to evaluate the contributions that histidine residues might play both in the catalytic activities of endonuclease V and in binding to nontarget DNA, the technique of oligonucleotide site directed mutagenesis was used to create mutations at each of the four histidine residues in the endonuclease V gene. Although none of the histidines were shown to be absolutely required for the pyrimidine dimer specific DNA glycosylase activity or the apurinic lyase activity, conservative amino acid changes at His16 produced enzymes with little or no catalytic activity. In addition, the evaluation of conservative and radical amino acid substitutions at positions 34, 56, and 107 is consistent with the interpretation that each of these histidines may be involved in nontarget DNA binding. The data supporting this conclusion are that histidine changes to lysine at positions 34 and 107 enhance the nontarget DNA binding activity of the mutant enzymes while neutralization of charge at His56 reduces nontarget DNA binding.
Numerous DNA-interactive proteins have been shown to locate specific sequences within large domains of non-target DNA in vitro and in vivo by a one-dimensional diffusion mechanism; however, the biological significance of this process has not been evaluated. We have examined the biological consequences of sliding for the pyrimidine dimer-specific DNA repair enzyme T4 endonuclease V, an enzyme which scans non-target DNA both in vitro and in vivo. An endonuclease V mutant was constructed whose only altered biochemical characteristic, measured in vitro, was a loss in its ability to slide on non-target DNA. In contrast to the native enzyme, when the mutated endonuclease V was expressed in DNA repair-deficient Escherichia coli, no enhanced ultraviolet survival was conferred. These results suggest that the mechanisms which DNA-interactive proteins employ to enhance the probability of locating their target sequences are of significant biological importance.
Endonuclease V, a pyrimidine dimer specific endonuclease in T4 bacteriophage, is able to scan DNA, recognize pyrimidine dimer photoproducts produced by exposure to ultraviolet light, and effectively incise DNA through a two-step mechanism at the damaged bases. The interaction of endonuclease V with nontarget DNA is thought to occur via electrostatic interactions between basic amino acids and the acidic phosphate DNA backbone. Arginine-3 was chosen as a potential candidate for involvement in this protein-nontarget DNA interaction and was extensively mutated to assess its role. The mutations include changes to Asp, Glu, Leu, and Lys and deleting it from the enzyme. Deletion of Arg-3 resulted in an enzyme that retained marginal levels of AP specificity, but no other detectable activity. Charge reversal to Glu-3 and Asp-3 results in proteins that exhibit AP-specific nicking and low levels of dimer-specific nicking. These enzymes are incapable of affecting cellular survival of repair-deficient Escherichia coli after irradiation. Mutations of Arg-3 to Lys-3 or Leu-3 also are unable to complement repair-deficient E. coli. However, these two proteins do exhibit a substantial level of in vitro dimer- and AP-specific nicking. The mechanism by which the Leu-3 and Lys-3 mutant enzymes locate pyrimidine dimers within a population of heavily irradiated plasmid DNA molecules appears to be significantly different from that for the wild-type enzyme. The wild-type endonuclease V processively incises all dimers on an individual plasmid prior to dissociation from that plasmid and subsequent reassociation with other plasmids, yet neither of these mutants exhibits any of the characteristics of this processive nicking activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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.
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.
The rate of depurination of double-stranded Bacillus subtilis DNA, radioactively labeled in the purine residues, has been followed as a function of temperature, pH, and ionic strength. In a Mg2+-containing buffer of physiological ionic strength, the rate constant for depurination of DNA is 4 × 10-9 sec-1 at 70° and pH 7.4. The activation energy of the reaction is 31 ± 2 kcal/mole. These data strongly indicate that depurination of DNA occurs at a physiologically significant rate under in vivo conditions and consequently that the lesions introduced in this fashion must be repaired.
Plasmids are double-stranded circular DNA molecules that have the property of self-replication, independent of chromosomal DNA. Although the presence of a plasmid in a bacterial cell may be detected genetically as a change in phenotype, often it is necessary to isolate plasmid DNA for molecular studies, such as size determination, restriction enzyme mapping, and nucleotide sequencing, or for the construction of new hybrid plasmids. The degree of purification required will depend upon the intended use. Less purified plasmid DNA is often satisfactory for recombinant DNA studies, and a large number of shorter and simpler methods have been developed. This chapter describes one such method that uses an alkaline extraction step. It is rapid enough to be used as a screening method, permitting 50-100 or more samples to be extracted in a few hours. The DNA is sufficiently pure to be digestible by restriction enzymes, an important advantage for screening. A preparative version that allows isolation of larger quantities of more highly purified material is also described.
A nuclear and a cytoplasmic uracil-DNA glycosylase have been purified from epithelial cells derived from a rat hepatoma (H4 cells) cultured in vitro. They have different optimum pH, molecular weight, isoelectric points, activation energy, Km. Uracil acts as a non competitive inhibitor towards the nuclear enzyme while it is a competitive one for the cytoplasmic enzyme. Comparison of the properties of the two mammalian enzymes with those of the enzymes isolated from Escherichia coli and Micrococcus luteus shows that they all behave differently. The following criteria were studied: molecular weight, optimum pH, isoelectric point, inhibition by uracil analogs, modulation of their activity by polyamines or by intercalating drugs. The only common properties shared by these four enzymes are: an activity twice as high on single stranded DNA than on double stranded DNA and no requirement for divalent cation for maximal activity.