Y-family DNA Polymerases Research Articles

Conformational Changes during Nucleotide Selection by Sulfolobus solfataricus DNA Polymerase Dpo4

The mechanism of nucleotide selection by Y-family DNA polymerases has been the subject of intense study, but significant structural contacts and/or conformational changes that relate to polymerase fidelity have been difficult to identify. Here we report on the conformational dynamics of a model Y-family polymerase Dpo4 from Sulfolobus solfataricus. Hydrogen-deuterium exchange in tandem with mass spectrometry was used to monitor changes in Dpo4 structure as a function of time and the presence or absence of specific substrates and ligands. Analysis of the data revealed previously unrecognized structural changes that accompany steps in the catalytic cycle leading up to phosphoryl transfer. For example, the solvent accessibility of the alphaB-loop-alphaC region in the finger domain decreased in the presence of all four dNTP insertion events, but the rate of deuterium exchange, an indicator of conformational flexibility, only decreased during an accurate insertion event. Of particular note is a change in the region surrounding the H-helix of the thumb domain. Upon binding DNA and Mg2+, the H-helix showed a decrease in solvent accessibility and flexibility that was relaxed only upon addition of dCTP, which forms a Watson-Crick base pair with template dG and not during mispairing events. The current study expands upon a previous report from our group that used a fluorescent probe located near the thumb domain to measure the kinetic properties of Dpo4 conformational changes. We now present a model for nucleotide selection by Dpo4 that arises from a synthesis of both structural and kinetic data.

Structure-Function Relationships in Miscoding by Sulfolobus solfataricus DNA Polymerase Dpo4: GUANINE N2,N2-DIMETHYL SUBSTITUTION PRODUCES INACTIVE AND MISCODING POLYMERASE COMPLEXES

Previous work has shown that Y-family DNA polymerases tolerate large DNA adducts, but a substantial decrease in catalytic efficiency and fidelity occurs during bypass of N2,N2-dimethyl (Me2)-substituted guanine (N2,N2-Me2G), in contrast to a single methyl substitution. Therefore, it is unclear why the addition of two methyl groups is so disruptive. The presence of N2,N2-Me2G lowered the catalytic efficiency of the model enzyme Sulfolobus solfataricus Dpo4 16,000-fold. Dpo4 inserted dNTPs almost at random during bypass of N2,N2-Me2G, and much of the enzyme was kinetically trapped by an inactive ternary complex when N2,N2-Me2G was present, as judged by a reduced burst amplitude (5% of total enzyme) and kinetic modeling. One crystal structure of Dpo4 with a primer having a 3'-terminal dideoxycytosine (Cdd) opposite template N2,N2-Me2G in a post-insertion position showed Cdd folded back into the minor groove, as a catalytically incompetent complex. A second crystal had two unique orientations for the primer terminal Cdd as follows: (i) flipped into the minor groove and (ii) a long pairing with N2,N2-Me2G in which one hydrogen bond exists between the O-2 atom of Cdd and the N-1 atom of N2,N2-Me2G, with a second water-mediated hydrogen bond between the N-3 atom of Cdd and the O-6 atom of N2,N2-Me2G. A crystal structure of Dpo4 with dTTP opposite template N2,N2-Me2G revealed a wobble orientation. Collectively, these results explain, in a detailed manner, the basis for the reduced efficiency and fidelity of Dpo4-catalyzed bypass of N2,N2-Me2G compared with mono-substituted N2-alkyl G adducts.

Structure of Human DNA Polymerase κ Inserting dATP Opposite an 8-OxoG DNA Lesion

BackgroundOxygen-free radicals formed during normal aerobic cellular metabolism attack bases in DNA and 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major lesions formed. It is amongst the most mutagenic lesions in cells because of its dual coding potential, wherein 8-oxoG(syn) can pair with an A in addition to normal base pairing of 8-oxoG(anti) with a C. Human DNA polymerase κ (Polκ) is a member of the newly discovered Y-family of DNA polymerases that possess the ability to replicate through DNA lesions. To understand the basis of Polκ's preference for insertion of an A opposite 8-oxoG lesion, we have solved the structure of Polκ in ternary complex with a template-primer presenting 8-oxoG in the active site and with dATP as the incoming nucleotide.Methodology and Principal FindingsWe show that the Polκ active site is well-adapted to accommodate 8-oxoG in the syn conformation. That is, the polymerase and the bound template-primer are almost identical in their conformations to that in the ternary complex with undamaged DNA. There is no steric hindrance to accommodating 8-oxoG in the syn conformation for Hoogsteen base-paring with incoming dATP.Conclusions and SignificanceThe structure we present here is the first for a eukaryotic translesion synthesis (TLS) DNA polymerase with an 8-oxoG:A base pair in the active site. The structure shows why Polκ is more efficient at inserting an A opposite the 8-oxoG lesion than a C. The structure also provides a basis for why Polκ is more efficient at inserting an A opposite the lesion than other Y-family DNA polymerases.

Structure-Function Relationships in Miscoding by Sulfolobus solfataricus DNA Polymerase Dpo4: GUANINE N2,N2-DIMETHYL SUBSTITUTION PRODUCES INACTIVE AND MISCODING POLYMERASE COMPLEXES

Structure-Function Relationships in Miscoding by Sulfolobus solfataricus DNA Polymerase Dpo4: GUANINE N2,N2-DIMETHYL SUBSTITUTION PRODUCES INACTIVE AND MISCODING POLYMERASE COMPLEXES

Structure of the Human Rev1–DNA–dNTP Ternary Complex

Y-family DNA polymerases have proven to be remarkably diverse in their functions and in strategies for replicating through DNA lesions. The structure of yeast Rev1 ternary complex has revealed the most radical replication strategy, where the polymerase itself dictates the identity of the incoming nucleotide, as well as the identity of the templating base. We show here that many of the key elements of this highly unusual strategy are conserved between yeast and human Rev1, including the eviction of template G from the DNA helix and the pairing of incoming deoxycytidine 5'-triphosphate with a surrogate arginine residue. We also show that the catalytic core of human Rev1 is uniquely augmented by two large inserts, I1 and I2, wherein I1 extends > 20 Å away from the active site and may serve as a platform for protein–protein interactions specific for Rev1's role in translesion DNA synthesis in human cells, and I2 acts as a “flap” on the hydrophobic pocket accommodating template G. We suggest that these novel structural features are important for providing human Rev1 greater latitude in promoting efficient and error-free translesion DNA synthesis through the diverse array of bulky and potentially carcinogenic N 2–deoxyguanosine DNA adducts in human cells.

Visualizing Sequence-Governed Nucleotide Selectivities and Mutagenic Consequences through a Replicative Cycle: Processing of a Bulky Carcinogen N2-dG Lesion in a Y-Family DNA Polymerase

Understanding how DNA polymerases process lesions remains fundamental to determining the molecular origins of mutagenic translesion bypass. We have investigated how a benzo[a]pyrene-derived N(2)-dG adduct, 10S-(+)-trans-anti-[BP]-N(2)-dG ([BP]G*), is processed in Dpo4, the well-characterized Y-family bypass DNA polymerase. This polymerase has a slippage-prone spacious active site region. Experimental results in a 5'-C[BP]G*G-3' sequence context reveal significant selectivity for dGTP insertion that predominantly yields -1 deletion extension products. A less pronounced error-prone nonslippage pathway that leads to full extension products with insertion of A > C > G opposite the lesion is also observed. Molecular modeling and dynamics simulations follow the bypass of [BP]G* through an entire replication cycle for the first time in Dpo4, providing structural interpretations for the experimental observations. The preference for dGTP insertion is explained by a 5'-slippage pattern in which the unmodified G rather than G* is skipped, the incoming dGTP pairs with the C on the 5'-side of G*, and the -1 deletion is produced upon further primer extension which is more facile than nucleotide insertion. In addition, the simulations suggest that the [BP]G* may undergo an anti/syn conformational rearrangement during the stages of the replication cycle. In the minor nonslippage pathway, the nucleotide insertion preferences opposite the lesion are explained by relative distortions to the active site region. These structural insights, provided by the modeling and dynamics studies, augment kinetic and limited available crystallographic investigations with bulky lesions, by providing molecular explanations for lesion bypass activities over an entire replication cycle.

Y-family DNA polymerases in mammalian cells.

Eukaryotic genomes are replicated with high fidelity to assure the faithful transmission of genetic information from one generation to the next. The accuracy of replication relies heavily on the ability of replicative DNA polymerases to efficiently select correct nucleotides for the polymerization reaction and, using their intrinsic exonuclease activities, to excise mistakenly incorporated nucleotides. Cells also possess a variety of specialized DNA polymerases that, by a process called translesion DNA synthesis (TLS), help overcome replication blocks when unrepaired DNA lesions stall the replication machinery. This review considers the properties of the Y-family (a subset of specialized DNA polymerases) and their roles in modulating spontaneous and genotoxic-induced mutations in mammals. We also review recent insights into the molecular mechanisms that regulate PCNA monoubiquitination and DNA polymerase switching during TLS and discuss the potential of using Y-family DNA polymerases as novel targets for cancer prevention and therapy.

Mechanistic Studies of the Bypass of a Bulky Single-base Lesion Catalyzed by a Y-family DNA Polymerase

1-nitropyrene, the most abundant nitro polycyclic aromatic hydrocarbon in diesel emissions, has been found to react with DNA to form predominantly N-(deoxyguanosin-8-yl)-1-aminopyrene (dGAP). This bulky adduct has been shown to induce genetic mutations, which may implicate Y-family DNA polymerases in its bypass in vivo. To establish a kinetic mechanism for the bypass of such a prototype single-base lesion, we employed pre-steady-state kinetic methods to investigate individual nucleotide incorporations upstream, opposite, and downstream from a site-specifically placed dGAP lesion catalyzed by Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase. Dpo4 was able to bypass dGAP but paused strongly at two sites: opposite the lesion and immediately downstream from the lesion. Both nucleotide incorporation efficiency and fidelity decreased significantly at the pause sites, especially during extension of the bypass product. Interestingly, a 4-fold tighter binding affinity of damaged DNA to Dpo4 promoted catalysis through putative interactions between the active site residues of Dpo4 and 1-aminopyrene moiety at the first pause site. In the presence of a DNA trap, the kinetics of nucleotide incorporation at these sites was biphasic in which a small, fast phase preceded a larger, slow phase. In contrast, only a large, fast phase was observed during nucleotide incorporation at non-pause sites. Our kinetic studies support a general kinetic mechanism for lesion bypass catalyzed by numerous DNA polymerases.

Penicilliols A and B, novel inhibitors specific to mammalian Y-family DNA polymerases

Penicilliols A ( 1) and B ( 2) are novel 5-methoxy-3(2 H)-furanones isolated from cultures of a fungus ( Penicillium daleae K.M. Zalessky) derived from a sea moss, and their structures were determined by spectroscopic analyses. These compounds selectively inhibited activities of eukaryotic Y-family DNA polymerases (pols) (i.e., pols η, ι and κ), and compound 1 was a stronger inhibitor than compound 2. Among mammalian Y-family pols, mouse pol ι activity was most strongly inhibited by compounds 1 and 2, with IC 50 values of 19.8 and 32.5 μM, respectively. On the other hand, activities of many other pols, such as A-family (i.e., pol γ), B-family (i.e., pols α, δ and ε) or X-family (i.e., pols β, λ and terminal deoxynucleotidyl transferase), and some DNA metabolic enzymes, such as calf primase of pol α, human immunodeficiency virus type-1 (HIV-1) reverse transcriptase, human telomerase, T7 RNA polymerase, mouse IMP dehydrogenase (type II), human topoisomerases I and II, T4 polynucleotide kinase or bovine deoxyribonuclease I, are not influenced by these compounds. In conclusion, this is the first report on potent inhibitors of mammalian Y-family pols.

DNA polymerase kappa from Trypanosoma cruzi localizes to the mitochondria, bypasses 8‐oxoguanine lesions and performs DNA synthesis in a recombination intermediate

DNA polymerase kappa (Pol kappa) is a low-fidelity polymerase that has the ability to bypass several types of lesions. The biological role of this enzyme, a member of the DinB subfamily of Y-family DNA polymerases, has remained elusive. In this report, we studied one of the two copies of Pol kappa from the protozoan Trypanosoma cruzi (TcPol kappa). The role of this TcPol kappa copy was investigated by analysing its subcellular localization, its activities in vitro, and performing experiments with parasites that overexpress this polymerase. The TcPOLK sequence has the N-terminal extension which is present only in eukaryotic DinB members, but its C-terminal region is more similar to prokaryotic and archaeal counterparts since it lacks C(2)HC motifs and PCNA interaction domain. Our results indicate that in contrast to its previously described orthologues, this polymerase is localized to mitochondria. The overexpression of TcPOLK increases T. cruzi resistance to hydrogen peroxide, and in vitro polymerization assays revealed that TcPol kappa efficiently bypasses 8-oxoguanine lesions. Remarkably, our results also demonstrate that the DinB subfamily of polymerases can participate in homologous recombination, based on our findings that TcPol kappa increases T. cruzi resistance to high doses of gamma irradiation and zeocin and can catalyse DNA synthesis within recombination intermediates.

Y-Family DNA polymerases may use two different dNTP shapes for insertion: A hypothesis and its implications

Chemicals and radiation can damage DNA leading to the formation of adducts/lesions, which – if not removed by DNA repair pathways – usually block replicative DNA polymerases (DNAPs). To overcome such potentially lethal blockage, cells have lesion bypass DNAPs, which are often in the Y-Family and include several classes. One class includes human DNAP κ and E. coli DNAP IV, and they insert dCTP in the non-mutagenic pathway opposite [+ta]-B[a]P-N 2-dG, which is the major adduct formed by the environmental carcinogen benzo[a]pyrene. Another class includes hDNAP η and ecDNAP V, and they insert dATP opposite [+ta]-B[a]P-N 2-dG in the dominant G → T mutagenic pathway. Herein we develop a hypothesis for why the IV/κ-class preferentially does cellular dCTP insertion. On the minor groove side of the active site, Y-Family DNAPs have a cleft/hole that can be analyzed based on an analogy to a “chimney.” Our models of DNAP IV show a large chimney opening from which the pyrene of [+ta]-B[a]P-N 2-dG can protrude, which allows canonical adduct-dG:dCTP pairing. In contrast, our models of DNAP V have small chimney openings that forces adduct-dG downward in the active site such that canonical adduct-dG:dCTP pairing is not possible. Based on X-ray structures, sequence alignment and our modeled structures of Y-Family DNAPs, chimney opening size seems primarily controlled by one amino acid (“flue-handle”), which dictates whether nearby amino acids (“flue”) plug the chimney or not. Based on this analysis, a correlation is apparent: the flue is closed in V/η-class DNAPs giving small chimney openings, while the flue is open for the IV/κ-class giving large chimney openings. Secondarily, a hypothesis is developed for why the V/η-class might preferentially do cellular dATP insertion opposite [+ta]-B[a]P-N 2-dG: the small chimney forces adduct-dG lower in the active site, possibly leading to catalysis using a non-canonical dNTP shape that permits syn-adenine:adduct-dG base pairing. In summary, a hypothesize is developed that the pyrene moiety of [+ta]-B[a]P-N 2-dG protrudes from the large chimney opening of DNAP IV, thus permitting canonical dCTP:adduct-dG pairing, while the small chimney opening of DNAP V forces [+ta]-B[a]P-N 2-dG lower down in the active site, in which syn-adenine can pair with adduct-dG via a non-canonical dNTP shape.

The N-clasp of human DNA polymerase kappa promotes blockage or error-free bypass of adenine- or guanine-benzo[a]pyrenyl lesions.

DNA bypass polymerases are utilized to transit bulky DNA lesions during replication, but the process frequently causes mutations. The structural origins of mutagenic versus high fidelity replication in lesion bypass is therefore of fundamental interest. As model systems, we investigated the molecular basis of the experimentally observed essentially faithful bypass of the guanine 10S-(+)-trans-anti-benzo[a]pyrene-N2-dG adduct by the Y-family human DNA polymerase κ, and the observed blockage of pol κ produced by the adenine 10S-(+)-trans-anti-benzo[a]pyrene-N2-dA adduct. These lesions are derived from the most tumorigenic metabolite of the ubiquitous cancer-causing pollutant, benzo[a]pyrene. We compare our results for the dG adduct with our earlier studies for the pol κ archaeal homolog Dpo4, which processes the same lesion in an error-prone manner. Molecular modeling, molecular mechanics calculations and molecular dynamics simulations were utilized. Our results show that the pol κ N-clasp is a key structural feature that accounts for the dA adduct blockage and the near-error-free bypass of the dG lesion. Absence of the N-clasp in Dpo4 explains the error-prone processing of the same lesion by this enzyme. Thus, our studies elucidate structure-function relationships in the fidelity of lesion bypass.

Initial crystallographic study of human PCNA in complex with a peptide containing the noncanonical PIP-box sequence of human DNA polymerase iota.

Human DNA polymerase iota (Poliota) is one of the Y-family DNA polymerases involved in translesion synthesis (TLS), which allows continued replication at damaged DNA templates. Poliota has a noncanonical PCNA-interacting protein box (PIP-box) within an internal region of the protein. Poliota activity is stimulated by PCNA binding through the noncanonical PIP-box. To clarify the interaction of PCNA with the noncanonical PIP-box of Poliota, PCNA and a Poliota peptide carrying the noncanonical PIP-box complex have been cocrystallized. The crystal belongs to space group C2, with unit-cell parameters a = 167.1, b = 68.7, c = 90.0 A, beta = 95.1 degrees . Structural analysis by molecular replacement is in progress.

RAD18 and associated proteins are immobilized in nuclear foci in human cells entering S-phase with ultraviolet light-induced damage

Proteins required for translesion DNA synthesis localize in nuclear foci of cells with replication-blocking lesions. The dynamics of this process were examined in human cells with fluorescence-based biophysical techniques. Photobleaching recovery and raster image correlation spectroscopy experiments indicated that involvement in the nuclear foci reduced the movement of RAD18 from diffusion-controlled to virtual immobility. Examination of the mobility of REV1 indicated that it is similarly immobilized when it is observed in nuclear foci. Reducing the level of RAD18 greatly reduced the focal accumulation of REV1 and reduced UV mutagenesis to background frequencies. Fluorescence lifetime measurements indicated that RAD18 and RAD6A or polη only transferred resonance energy when these proteins colocalized in damage-induced nuclear foci, indicating a close physical association only within such foci. Our data support a model in which RAD18 within damage-induced nuclear foci is immobilized and is required for recruitment of Y-family DNA polymerases and subsequent mutagenesis. In the absence of damage these proteins are not physically associated within the nucleoplasm.

Effect of Proliferating Cell Nuclear Antigen Ubiquitination and Chromatin Structure on the Dynamic Properties of the Y-family DNA Polymerases

Y-family DNA polymerases carry out translesion synthesis past damaged DNA. DNA polymerases (pol) eta and iota are usually uniformly distributed through the nucleus but accumulate in replication foci during S phase. DNA-damaging treatments result in an increase in S phase cells containing polymerase foci. Using photobleaching techniques, we show that poleta is highly mobile in human fibroblasts. Even when localized in replication foci, it is only transiently immobilized. Although ubiquitination of proliferating cell nuclear antigen (PCNA) is not required for the localization of poleta in foci, it results in an increased residence time in foci. poliota is even more mobile than poleta, both when uniformly distributed and when localized in foci. Kinetic modeling suggests that both poleta and poliota diffuse through the cell but that they are transiently immobilized for approximately 150 ms, with a larger proportion of poleta than poliota immobilized at any time. Treatment of cells with DRAQ5, which results in temporary opening of the chromatin structure, causes a dramatic immobilization of poleta but not poliota. Our data are consistent with a model in which the polymerases are transiently probing the DNA/chromatin. When DNA is exposed at replication forks, the polymerase residence times increase, and this is further facilitated by the ubiquitination of PCNA.

Quantum mechanics/molecular mechanics investigation of the chemical reaction in Dpo4 reveals water-dependent pathways and requirements for active site reorganization.

The nucleotidyl-transfer reaction coupled with the conformational transitions in DNA polymerases is critical for maintaining the fidelity and efficiency of DNA synthesis. We examine here the possible reaction pathways of a Y-family DNA polymerase, Sulfolobus solfataricus DNA polymerase IV (Dpo4), for the correct insertion of dCTP opposite 8-oxoguanine using the quantum mechanics/molecular mechanics (QM/MM) approach, both from a chemistry-competent state and a crystal closed state. The latter examination is important for understanding pre-chemistry barriers to interpret the entire enzyme mechanism, since the crystal closed state is not an ideal state for initiating the chemical reaction. The most favorable reaction path involves initial deprotonation of O3'H via two bridging water molecules to O1A, overcoming an overall potential energy barrier of approximately 20.0 kcal/mol. The proton on O1A-P(alpha) then migrates to the gamma-phosphate oxygen of the incoming nucleotide as O3' attacks P(alpha), and the P(alpha)-O3A bond breaks. The other possible pathway in which the O3'H proton is transferred directly to O1A on P(alpha) has an overall energy barrier of 25.0 kcal/mol. In both reaction paths, the rate-limiting step is the initial deprotonation, and the trigonal-bipyramidal configuration for P(alpha) occurs during the concerted bond formation (O3'-P(alpha)) and breaking (P(alpha)-O3A), indicating the associative nature of the chemical reaction. In contrast, the Dpo4/DNA complex with an imperfect active-site geometry corresponding to the crystal state must overcome a much higher activation energy barrier (29.0 kcal/mol) to achieve a tightly organized site due to hindered O3'H deprotonation stemming from larger distances and distorted conformation of the proton acceptors. This significant difference demonstrates that the pre-chemistry reorganization in Dpo4 costs approximately 4.0 to 9.0 kcal/mol depending on the primer terminus environment. Compared to the higher fidelity DNA polymerase beta from the X-family, Dpo4 has a higher chemical reaction barrier (20.0 vs 15.0 kcal/mol) due to the more solvent-exposed active site.

Mutational Specificity of γ-Radiation-Induced Guanine−Thymine and Thymine−Guanine Intrastrand Cross-Links in Mammalian Cells and Translesion Synthesis Past the Guanine−Thymine Lesion by Human DNA Polymerase η

Comparative mutagenesis of γ- or X-ray-induced tandem DNA lesions G[8,5-Me]T and T[5-Me,8]G intrastrand cross-links was investigated in simian (COS-7) and human embryonic (293T) kidney cells. For G[8,5-Me]T in 293T cells, 5.8% of progeny contained targeted base substitutions, whereas 10.0% showed semitargeted single-base substitutions. Of the targeted mutations, the G → T mutation occurred with the highest frequency. The semitargeted mutations were detected up to two bases 5′ and three bases 3′ to the cross-link. The most prevalent semitargeted mutation was a C → T transition immediately 5′ to the G[8,5-Me]T cross-link. Frameshifts (4.6%) (mostly small deletions) and multiple-base substitutions (2.7%) also were detected. For the T[5-Me,8]G cross-link, a similar pattern of mutations was noted, but the mutational frequency was significantly higher than that of G[8,5-Me]T. Both targeted and semitargeted mutations occurred with a frequency of ∼16%, and both included a dominant G → T transversion. As in 293T cells, more than twice as many targeted mutations in COS cells occurred in T[5-Me,8]G (11.4%) as in G[8,5-Me]T (4.7%). Also, the level of semitargeted single-base substitutions 5′ to the lesion was increased and 3′ to the lesion decreased in T[5-Me,8]G relative to G[8,5-Me]T in COS cells. It appeared that the majority of the base substitutions at or near the cross-links resulted from incorporation of dAMP opposite the template base, in agreement with the so-called “A-rule”. To determine if human polymerase η (hpol η) might be involved in the mutagenic bypass, an in vitro bypass study of G[8,5-Me]T in the same sequence was carried out, which showed that hpol η can bypass the cross-link incorporating the correct dNMP opposite each cross-linked base. For G[8,5-Me]T, nucleotide incorporation by hpol η was significantly different from that by yeast pol η in that the latter was more error-prone opposite the cross-linked Gua. The incorporation of the correct nucleotide, dAMP, by hpol η opposite cross-linked T was 3−5-fold more efficient than that of a wrong nucleotide, whereas incorporation of dCMP opposite the cross-linked G was 10-fold more efficient than that with dTMP. Therefore, the nucleotide incorporation pattern by hpol η was not consistent with the observed cellular mutations. Nevertheless, at and near the lesion, hpol η was more error-prone compared to a control template. The in vitro data suggest that translesion synthesis by another Y-family DNA polymerase and/or flawed participation of an accessory protein is a more likely scenario in the mutagenesis of these lesions in mammalian cells. However, hpol η may play a role in correct bypass of the cross-links.

Efficient and accurate bypass of N 2 -(1-carboxyethyl)-2′-deoxyguanosine by DinB DNA polymerase in vitro and in vivo

DinB, a Y-family DNA polymerase, is conserved among all domains of life; however, its endogenous substrates have not been identified. DinB is known to synthesize accurately across a number of N(2)-dG lesions. Methylglyoxal (MG) is a common byproduct of the ubiquitous glycolysis pathway and induces the formation of N(2)-(1-carboxyethyl)-2'-deoxyguanosine (N(2)-CEdG) as the major stable DNA adduct. Here, we found that N(2)-CEdG could be detected at a frequency of one lesion per 10(7) nucleosides in WM-266-4 human melanoma cells, and treatment of these cells with MG or glucose led to a dose-responsive increase in N(2)-CEdG formation. We further constructed single-stranded M13 shuttle vectors harboring individual diastereomers of N(2)-CEdG at a specific site and assessed the cytotoxic and mutagenic properties of the lesion in wild-type and bypass polymerase-deficient Escherichia coli cells. Our results revealed that N(2)-CEdG is weakly mutagenic, and DinB (i.e., polymerase IV) is the major DNA polymerase responsible for bypassing the lesion in vivo. Moreover, steady-state kinetic measurements showed that nucleotide insertion, catalyzed by E. coli pol IV or its human counterpart (i.e., polymerase kappa), opposite the N(2)-CEdG is both accurate and efficient. Taken together, our data support that N(2)-CEdG, a minor-groove DNA adduct arising from MG, is an important endogenous substrate for DinB DNA polymerase.

Miscoding Properties of 6α- and 6β-Diastereoisomers of the N2-(Estradiol-6-yl)-2′-deoxyguanosine DNA Adduct by Y-Family Human DNA Polymerases

Treatment with estrogen increases the risk of breast, ovary, and endometrial cancers in women. DNA damage induced by estrogen is thought to be involved in estrogen carcinogenesis. In fact, Y-family human DNA polymerases (pol) eta and kappa, which are highly expressed in the reproductive organs, miscode model estrogen-derived DNA adducts during DNA synthesis. Since the estrogen-DNA adducts are a mixture of 6alpha- and 6beta-diastereoisomers of dG-N(2)-6-estrogen or dA-N(6)-6-estrogen, the stereochemistry of each isomeric adduct on translesion synthesis catalyzed by DNA pols has not been investigated. We have recently established a phosphoramidite chemical procedure to insert 6alpha- or 6beta-isomeric N(2)-(estradiol-6-yl)-2'-deoxyguanosine (dG-N(2)-6-E(2)) into oligodeoxynucleotides. Using such site-specific modified oligomer as a template, the specificity and frequency of miscoding by dG-N(2)-6alpha-E(2) or dG-N(2)-6beta-E(2) were explored using pol eta and a truncated form of pol kappa (pol kappaDeltaC). Translesion synthesis catalyzed by pol eta bypassed both the 6alpha- and 6beta-isomers of dG-N(2)-6-E(2), with a weak blockage at the adduct site, while translesion synthesis catalyzed by pol kappaDeltaC readily bypassed both isomeric adducts. Quantitative analysis of base substitutions and deletions occurring at the adduct site showed that pol kappaDeltaC was more efficient than pol eta by incorporating dCMP opposite both 6alpha- and 6beta-isomeric dG-N(2)-6-E(2) adducts. The miscoding events occurred more frequently with pol eta, but not with pol kappaDeltaC. Pol eta promoted incorporation of dAMP and dTMP at both the 6alpha- and 6beta-isomeric adducts, generating G --> T transversions and G --> A transitions. One- and two-base deletions were also formed. The 6alpha-isomeric adduct promoted slightly lower frequency of dCMP incorporation and higher frequency of dTMP incorporation and one-base deletions, compared with the 6beta-isomeric adduct. These observations were supported by steady-state kinetic studies. Taken together, the miscoding property of the 6alpha-isomeric dG-N(2)-6-E(2) is likely to be similar to that of the 6beta-isomeric adduct.

Mechanism of double-base lesion bypass catalyzed by a Y-family DNA polymerase

As a widely used anticancer drug, cis-diamminedichloroplatinum(II) (cisplatin) reacts with adjacent purine bases in DNA to form predominantly cis-[Pt(NH3)2{d(GpG)-N7(1),-N7(2)}] intrastrand cross-links. Drug resistance, one of the major limitations of cisplatin therapy, is partially due to the inherent ability of human Y-family DNA polymerases to perform translesion synthesis in the presence of DNA-distorting damage such as cisplatin–DNA adducts. To better understand the mechanistic basis of translesion synthesis contributing to cisplatin resistance, this study investigated the bypass of a single, site-specifically placed cisplatin-d(GpG) adduct by a model Y-family DNA polymerase, Sulfolobus solfataricus DNA polymerase IV (Dpo4). Dpo4 was able to bypass this double-base lesion, although, the incorporation efficiency of dCTP opposite the first and second cross-linked guanine bases was decreased by 72- and 860-fold, respectively. Moreover, the fidelity at the lesion decreased up to two orders of magnitude. The cisplatin-d(GpG) adduct affected six downstream nucleotide incorporations, but interestingly the fidelity was essentially unaltered. Biphasic kinetic analysis supported a universal kinetic mechanism for the bypass of DNA lesions catalyzed by various translesion DNA polymerases. In conclusion, if human Y-family DNA polymerases adhere to this bypass mechanism, then translesion synthesis by these error-prone enzymes is likely accountable for cisplatin resistance observed in cancer patients.