Nucleotide Insertion Opposite Research Articles

Structural Basis for Human DNA Polymerase Kappa to Bypass Cisplatin Intrastrand Cross-Link (Pt-GG) Lesion as an Efficient and Accurate Extender

Cisplatin (cis-diamminedichloroplatinum) is a common chemotherapeutic drug that reacts with the N7 atoms of adjacent guanines in DNA to form the Pt-1,2-d(GpG) intrastrand cross-link (Pt-GG), a major product to block DNA replication. Translesion DNA synthesis has been implicated in chemoresistance during cisplatin treatment of cancer due to Pt-GG lesion bypass. Gene knockdown studies in human cells have indicated a role for polκ during translesion synthesis of the Pt-GG lesion. However, the bypass activity of polκ with cisplatin lesions has not been well characterized. In this study, we investigated polκ's ability to bypass Pt-GG lesion in vitro and determined two crystal structures of polκ in complex with Pt-GG DNA. The ternary complex structures represent two consecutive stages of lesion bypass: nucleotide insertion opposite the 5′G (Pt-GG2) and primer extension immediately after the lesion (Pt-GG3). Our biochemical data showed that polκ is very efficient and accurate in extending DNA primers after the first G of the Pt-GG lesion. The structures demonstrate that the efficiency and accuracy is achieved by stably accommodating the bases with the cisplatin adduct in the active site for proper Watson–Crick base pairing with the incoming nucleotide in both the second insertion and post-insertion complexes. Our studies suggest that polκ works as an extender for efficient replication of the Pt-GG lesion in cells. This work holds promise for considering polκ, along with polη, as potential targets for drug design, which together could improve the efficacy of cisplatin treatment for cancer therapy.

Structural basis for proficient nucleotide incorporation across a major cisplatin‐DNA lesion by human DNA polymerase kappa (polκ)

Cisplatin (cis‐diamminedichloroplatinum) is a widely used chemotherapeutic compound. It reacts with the N7 atoms of adjacent guanines in DNA to form the Pt‐1,2‐d(GpG) intrastrand cross‐link (Pt‐GG) as a major product. This bulky cisplatin adduct blocks DNA replication by replicative polymerases, thereby promoting apoptosis of rapidly dividing cancer cells. However, cancer cells have developed resistance to cisplatin, partly because of the bypass of cisplatin adducts by specialized DNA polymerases (mostly belonging to the Y‐family) through translesion synthesis (TLS). The human Y‐family DNA polymerase eta (polη) is very efficient in replicating opposite the Pt‐GG lesions, but not in extending after this adduct. Recently, it was shown that human Y‐family DNA polymerase kappa (polκ) is indispensable for DNA repair synthesis in dorsal root ganglion neurons exposed to cisplatin. Polκ gene knockdown experiments in human cells have also indicated the involvement of polκ in TLS of the Pt‐GG lesion. In this study, we investigated the bypass potential and nucleotide incorporation preference of polκ opposite DNA containing the Pt‐GG adduct and determined two crystal structures of polκ complexed with such DNA. The ternary complex structures represent two consecutive stages of lesion bypass: nucleotide insertion opposite the 5′G (Pt‐GG2) and primer extension immediately after the lesion (Pt‐GG3). Our in vitro DNA replication assays using polκ and DNA containing the Pt‐GG lesion indicate that polκ inserts the correct dCTP opposite the 5′G of Pt‐GG (Pt‐GG2), when a nucleotide is already inserted opposite the 3′G (Pt‐GG1). Polκ also inserts the correct dATP opposite the T base immediately after the Pt‐GG (Pt‐GG3). However, polκ is not able to insert a nucleotide opposite the 3′G of Pt‐GG (Pt‐GG1). On the other hand, polη is very efficient in replicating the Pt‐GG lesion at two insertion stages (Pt‐GG1 and Pt‐GG2), but the enzyme is blocked at the extension stage (Pt‐GG3). In terms of fidelity, we found that polκ has the fewest mis‐insertions compared to two other human Y‐family polymerases polη and polι. Our steady‐state kinetic analysis showed that polκ incorporates dCTP and dATP opposite the 5′G of Pt‐GG (Pt‐GG2) and T template 5′ to the lesion (Pt‐GG3), respectively, with very similar relative efficiencies of 0.15–0.20, which are only ~5 fold lower than for normal DNA replication (GG2 and GG3). Our biochemical data indicate that polκ is very proficient and accurate in inserting a nucleotide after the first G of the Pt‐GG lesion. The structures revealed that the accuracy is achieved by stably accommodating the bases containing the cisplatin adducts in the enzyme's active site for proper Watson‐Crick base pairing with the incoming nucleotide. Together, our biochemical and structural data provide the molecular basis for the accurate and proficient incorporation by polκ of nucleotides opposite the cisplatin adduct and suggest an important role of polκ as a second polymerase (extender) in the efficient bypass of the Pt‐GG lesion in vivo. This work holds promise for polκ as a potential target for drug design, along with polη, which together could improve the efficacy of cisplatin treatment for cancer therapy.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Six Germline Genetic Variations Impair the Translesion Synthesis Activity of Human DNA Polymerase κ.

DNA polymerase (pol) κ efficiently catalyzes error-free translesion DNA synthesis (TLS) opposite bulky N2-guanyl lesions induced by carcinogens such as polycyclic aromatic hydrocarbons. We investigated the biochemical effects of nine human nonsynonymous germline POLK variations on the TLS properties of pol κ, utilizing recombinant pol κ (residues 1-526) enzymes and DNA templates containing an N2-CH2(9-anthracenyl)G (N2-AnthG), 8-oxo-7,8-dihydroguanine (8-oxoG), O6-methyl(Me)G, or an abasic site. In steady-state kinetic analyses, the R246X, R298H, T473A, and R512W variants displayed 7- to 18-fold decreases in kcat/Km for dCTP insertion opposite G and N2-AnthG, with 2- to 3-fold decreases in DNA binding affinity, compared to that of the wild-type, and further showed 5- to 190-fold decreases in kcat/Km for next-base extension from C paired with N2-AnthG. The A471V variant showed 2- to 4-fold decreases in kcat/Km for correct nucleotide insertion opposite and beyond G (or N2-AnthG) compared to that of the wild-type. These five hypoactive variants also showed similar patterns of attenuation of TLS activity opposite 8-oxoG, O6-MeG, and abasic lesions. By contrast, the T44M variant exhibited 7- to 11-fold decreases in kcat/Km for dCTP insertion opposite N2-AnthG and O6-MeG (as well as for dATP insertion opposite an abasic site) but not opposite both G and 8-oxoG, nor beyond N2-AnthG, compared to that of the wild-type. These results suggest that the R246X, R298H, T473A, R512W, and A471V variants cause a general catalytic impairment of pol κ opposite G and all four lesions, whereas the T44M variant induces opposite lesion-dependent catalytic impairment, i.e., only opposite O6-MeG, abasic, and bulky N2-G lesions but not opposite G and 8-oxoG, in pol κ, which might indicate that these hypoactive pol κ variants are genetic factors in modifying individual susceptibility to genotoxic carcinogens in certain subsets of populations.

Structure and mechanism of error-free replication past the major benzo[a]pyrene adduct by human DNA polymerase κ.

Benzo[a]pyrene (BP) is a well-known and frequently encountered carcinogen which generates a bulky DNA adduct (+)-trans-10S-BP-N2-dG (BP-dG) in cells. DNA polymerase kappa (polκ) is the only known Y-family polymerase that bypasses BP-dG accurately and thus protects cells from genotoxic BP. Here, we report the structures of human polκ in complex with DNA containing either a normal guanine (G) base or a BP-dG adduct at the active site and a correct deoxycytidine. The structures and supporting biochemical data reveal a unique mechanism for accurate replication by translesion synthesis past the major bulky adduct. The active site of polκ opens at the minor groove side of the DNA substrate to accommodate the bulky BP-dG that is attached there. More importantly, polκ stabilizes the lesion DNA substrate in the same active conformation as for regular B-form DNA substrates and the bulky BPDE ring in a 5′ end pointing conformation. The BP-dG adducted DNA substrate maintains a Watson–Crick (BP-dG:dC) base pair within the active site, governing correct nucleotide insertion opposite the bulky adduct. In addition, polκ's unique N-clasp domain supports the open conformation of the enzyme and the extended conformation of the single-stranded template to allow bypass of the bulky lesion. This work illustrates the first molecular mechanism for how a bulky major adduct is replicated accurately without strand misalignment and mis-insertion.

Effects of the N terminus of mouse DNA polymerase κ on the bypass of a guanine-benzo[a]pyrenyl adduct

DNA polymerase κ (Polκ), one of the typical member of the Y-family DNA polymerases, has been demonstrated to bypass the 10S(+)-trans-anti-benzo[a]pyrene diol epoxide-N(2)-deoxyguanine adducts (BPDE-dG) efficiently and accurately. A large structural gap between the core and little finger as well as an N-clasp domain are essential to its unique translesion capability. However, whether the extreme N-terminus of Polκ is required for its activity is unclear. In this work, we constructed two mouse Polκ deletions, which have either a catalytic core (mPolκ1-516) or a core without the first 21-residues (mPolκ22-516), and tested their activities in the replication of normal and BPDE-DNA. These two Polκ deletions are nearly as efficient as the full length protein (Polκ1-852) in normal DNA synthesis. However, steady-state kinetics reveals a significant reduction in efficiency of dCTP incorporation opposite the lesion by Polκ22-516, along with increased frequencies for misinsertion compared with Polκ1-852 The next nucleotide insertion opposite the template C immediately following the BPDE-dG was also examined, and the bypass differences induced by deletions were highlighted in both insertion and extension step. We conclude that the extreme N-terminal part of Polκ is required for the processivity and fidelity of Polκ during translesion synthesis of BPDE-dG lesions.

Guanine oxidation product 5-carboxamido-5-formamido-2-iminohydantoin induces mutations when bypassed by DNA polymerases and is a substrate for base excision repair.

Guanine (G) is a target for oxidation by reactive oxygen species in DNA, RNA, and the nucleotide pool. Damage to DNA yields products with alternative properties toward DNA processing enzymes compared to those of the parent nucleotide. A new lesion, 5-carboxamido-5-formamido-2-iminohydantoin (2Ih), bearing a stereocenter in the base was recently identified from the oxidation of G. DNA polymerase and base excision repair processing of this new lesion has now been evaluated. Single nucleotide insertion opposite (S)-2Ih and (R)-2Ih in the template strand catalyzed by the DNA polymerases Klenow fragment exo(-), DPO4, and Hemo KlenTaq demonstrates these lesions to cause point mutations. Specifically, they promote 3-fold more G·C → C·G transversion mutations than G·C → T·A, and (S)-2Ih was 2-fold more blocking for polymerase bypass than (R)-2Ih. Both diastereomer lesions were found to be substrates for the DNA glycosylases NEIL1 and Fpg, and poorly excised by endonuclease III (Nth). The activity was independent of the base pair partner. Thermal melting, CD spectroscopy, and density functional theory geometric optimization calculations were conducted to provide insight into these polymerase and DNA glycosylase studies. These results identify that formation of the 2Ih lesions in a cell would be mutagenic in the event that they were not properly repaired.

DNA polymerase ζ-dependent lesion bypass in Saccharomyces cerevisiae is accompanied by error-prone copying of long stretches of adjacent DNA.

Translesion synthesis (TLS) helps cells to accomplish chromosomal replication in the presence of unrepaired DNA lesions. In eukaryotes, the bypass of most lesions involves a nucleotide insertion opposite the lesion by either a replicative or a specialized DNA polymerase, followed by extension of the resulting distorted primer terminus by DNA polymerase ζ (Polζ). The subsequent events leading to disengagement of the error-prone Polζ from the primer terminus and its replacement with an accurate replicative DNA polymerase remain largely unknown. As a first step toward understanding these events, we aimed to determine the length of DNA stretches synthesized in an error-prone manner during the Polζ-dependent lesion bypass. We developed new in vivo assays to identify the products of mutagenic TLS through a plasmid-borne tetrahydrofuran lesion and a UV-induced chromosomal lesion. We then surveyed the region downstream of the lesion site (in respect to the direction of TLS) for the presence of mutations indicative of an error-prone polymerase activity. The bypass of both lesions was associated with an approximately 300,000-fold increase in the mutation rate in the adjacent DNA segment, in comparison to the mutation rate during normal replication. The hypermutated tract extended 200 bp from the lesion in the plasmid-based assay and as far as 1 kb from the lesion in the chromosome-based assay. The mutation rate in this region was similar to the rate of errors produced by purified Polζ during copying of undamaged DNA in vitro. Further, no mutations downstream of the lesion were observed in rare TLS products recovered from Polζ-deficient cells. This led us to conclude that error-prone Polζ synthesis continues for several hundred nucleotides after the lesion bypass is completed. These results provide insight into the late steps of TLS and show that error-prone TLS tracts span a substantially larger region than previously appreciated.

Replicative bypass of abasic site in Escherichia coli and human cells: similarities and differences.

Abasic [apurinic/apyrimidinic (AP)] sites are the most common DNA damages, opposite which dAMP is frequently inserted (‘A-rule’) in Escherichia coli. Nucleotide insertion opposite the AP-site in eukaryotic cells depends on the assay system and the type of cells. Accordingly, a ‘C-rule’, ‘A-rule’, or the lack of specificity has been reported. DNA sequence context also modulates nucleotide insertion opposite AP-site. Herein, we have compared replication of tetrahydrofuran (Z), a stable analog of AP-site, in E. coli and human embryonic kidney 293T cells in two different sequences. The efficiency of translesion synthesis or viability of the AP-site construct in E. coli was less than 1%, but it was 7- to 8-fold higher in the GZGTC sequence than in the GTGZC sequence. The difference in viability increased even more in pol V-deficient strains. Targeted one-base deletions occurred in 63% frequency in the GZG and 68% frequency in GZC sequence, which dropped to 49% and 21%, respectively, upon induction of SOS. The full-length products with SOS primarily involved dAMP insertion opposite the AP-site, which occurred in 49% and 71% frequency, respectively, in the GZG and GZC sequence. dAMP insertion, largely carried out by pol V, was more efficient when the AP-site was a stronger replication block. In contrast to these results in E. coli, viability was 2 to 3 orders of magnitude higher in human cells, and the ‘A-rule’ was more rigidly followed. The AP-site in the GZG and GZC sequences gave 76% and 89%, respectively, Z→T substitutions. In human cells, targeted one-base deletion was undetectable, and dTMP>dCMP were the next preferred nucleotides inserted opposite Z. siRNA knockdown of Rev1 or pol ζ established that both these polymerases are vital for AP-site bypass, as demonstrated by 36–67% reduction in bypass efficiency. However, neither polymerase was indispensable, suggesting roles of additional DNA polymerases in AP-site bypass in human cells.

Translesion Synthesis of 8,5′-Cyclopurine-2′-deoxynucleosides by DNA Polymerases η, ι, and ζ

Reactive oxygen species can give rise to a battery of DNA damage products including the 8,5'-cyclo-2'-deoxyadenosine (cdA) and 8,5'-cyclo-2'-deoxyguanosine (cdG) tandem lesions. The 8,5'-cyclopurine-2'-deoxynucleosides are quite stable lesions and are valid and reliable markers of oxidative DNA damage. However, it remains unclear how these lesions compromise DNA replication in mammalian cells. Previous in vitro biochemical assays have suggested a role for human polymerase (Pol) η in the insertion step of translesion synthesis (TLS) across the (5'S) diastereomers of cdA and cdG. Using in vitro steady-state kinetic assay, herein we showed that human Pol ι and a two-subunit yeast Pol ζ complex (REV3/REV7) could function efficiently in the insertion and extension steps, respectively, of TLS across S-cdA and S-cdG; human Pol κ and Pol η could also extend past these lesions, albeit much less efficiently. Results from a quantitative TLS assay showed that, in human cells, S-cdA and S-cdG inhibited strongly DNA replication and induced substantial frequencies of mutations at the lesion sites. Additionally, Pol η, Pol ι, and Pol ζ, but not Pol κ, had important roles in promoting replication through S-cdA and S-cdG in human cells. Based on these results, we propose a model for TLS across S-cdA and S-cdG in human cells, where Pol η and/or Pol ι carries out nucleotide insertion opposite the lesion, whereas Pol ζ executes the extension step.

Effects of N2-Alkylguanine, O6-Alkylguanine, and Abasic Lesions on DNA Binding and Bypass Synthesis by the Euryarchaeal B-Family DNA Polymerase Vent (exo–)

Archaeal and eukaryotic B-family DNA polymerases (pols) mainly replicate chromosomal DNA but stall at lesions, which are often bypassed with Y-family pols. In this study, a B-family pol Vent (exo(-)) from the euryarchaeon Thermococcus litoralis was studied with three types of DNA lesions-N(2)-alkylG, O(6)-alkylG, and an abasic (AP) site-in comparison with a model Y-family pol Dpo4 from Sulfolobus solfataricus, to better understand the effects of various DNA modifications on binding, bypass efficiency, and fidelity of pols. Vent (exo(-)) readily bypassed N(2)-methyl(Me)G and O(6)-MeG, but was strongly blocked at O(6)-benzyl(Bz)G and N(2)-BzG, whereas Dpo4 efficiently bypassed N(2)-MeG and N(2)-BzG and partially bypassed O(6)-MeG and O(6)-BzG. Vent (exo(-)) bypassed an AP site to an extent greater than Dpo4, corresponding with steady-state kinetic data. Vent (exo(-)) showed ~110-, 180-, and 300-fold decreases in catalytic efficiency (k(cat)/K(m)) for nucleotide insertion opposite an AP site, N(2)-MeG, and O(6)-MeG but ~1800- and 5000-fold decreases opposite O(6)-BzG and N(2)-BzG, respectively, as compared to G, whereas Dpo4 showed little or only ~13-fold decreases opposite N(2)-MeG and N(2)-BzG but ~260-370-fold decreases opposite O(6)-MeG, O(6)-BzG, and the AP site. Vent (exo(-)) preferentially misinserted G opposite N(2)-MeG, T opposite O(6)-MeG, and A opposite an AP site and N(2)-BzG, while Dpo4 favored correct C insertion opposite those lesions. Vent (exo(-)) and Dpo4 both bound modified DNAs with affinities similar to unmodified DNA. Our results indicate that Vent (exo(-)) is as or more efficient as Dpo4 in synthesis opposite O(6)-MeG and AP lesions, whereas Dpo4 is much or more efficient opposite (only) N(2)-alkylGs than Vent (exo(-)), irrespective of DNA-binding affinity. Our data also suggest that Vent (exo(-)) accepts nonbulky DNA lesions (e.g., N(2)- or O(6)-MeG and an AP site) as manageable substrates despite causing error-prone synthesis, whereas Dpo4 strongly favors minor-groove N(2)-alkylG lesions over major-groove or noninstructive lesions.

Chemistry and Biology of DNA Containing 1,N2-Deoxyguanosine Adducts of the α,β-Unsaturated Aldehydes Acrolein, Crotonaldehyde, and 4-Hydroxynonenal

The α,β-unsaturated aldehydes (enals) acrolein, crotonaldehyde, and trans-4-hydroxynonenal (4-HNE) are products of endogenous lipid peroxidation, arising as a consequence of oxidative stress. The addition of enals to dG involves Michael addition of the N2-amine to give N2-(3-oxopropyl)-dG adducts, followed by reversible cyclization of N1 with the aldehyde, yielding 1,N2-dG exocyclic products. The 1,N2-dG exocyclic adducts from acrolein, crotonaldehyde, and 4-HNE exist in human and rodent DNA. The enal-induced 1,N2-dG lesions are repaired by the nucleotide excision repair pathway in both Escherichia coli and mammalian cells. Oligodeoxynucleotides containing structurally defined 1,N2-dG adducts of acrolein, crotonaldehyde, and 4-HNE were synthesized via a postsynthetic modification strategy. Site-specific mutagenesis of enal adducts has been carried out in E. coli and various mammalian cells. In all cases, the predominant mutations observed are G→T transversions, but these adducts are not strongly miscoding. When placed into duplex DNA opposite dC, the 1,N2-dG exocyclic lesions undergo ring opening to the corresponding N2-(3-oxopropyl)-dG derivatives. Significantly, this places a reactive aldehyde in the minor groove of DNA, and the adducted base possesses a modestly perturbed Watson−Crick face. Replication bypass studies in vitro indicate that DNA synthesis past the ring-opened lesions can be catalyzed by pol η, pol ι, and pol κ. It also can be accomplished by a combination of Rev1 and pol ζ acting sequentially. However, efficient nucleotide insertion opposite the 1,N2-dG ring-closed adducts can be carried out only by pol ι and Rev1, two DNA polymerases that do not rely on the Watson−Crick pairing to recognize the template base. The N2-(3-oxopropyl)-dG adducts can undergo further chemistry, forming interstrand DNA cross-links in the 5′-CpG-3′ sequence, intrastrand DNA cross-links, or DNA−protein conjugates. NMR and mass spectrometric analyses indicate that the DNA interstand cross-links contain a mixture of carbinolamine and Schiff base, with the carbinolamine forms of the linkages predominating in duplex DNA. The reduced derivatives of the enal-mediated N2-dG:N2-dG interstrand cross-links can be processed in mammalian cells by a mechanism not requiring homologous recombination. Mutations are rarely generated during processing of these cross-links. In contrast, the reduced acrolein-mediated N2-dG peptide conjugates can be more mutagenic than the corresponding monoadduct. DNA polymerases of the DinB family, pol IV in E. coli and pol κ in human, are implicated in error-free bypass of model acrolein-mediated N2-dG secondary adducts, the interstrand cross-links, and the peptide conjugates.

Increased Flexibility Enhances Misincorporation: TEMPERATURE EFFECTS ON NUCLEOTIDE INCORPORATION OPPOSITE A BULKY CARCINOGEN-DNA ADDUCT BY A Y-FAMILY DNA POLYMERASE

The Y-family DNA polymerase Dpo4, from the thermophilic crenarchaeon Sulfolobus solfataricus P2, offers a valuable opportunity to investigate the effect of conformational flexibility on the bypass of bulky lesions because of its ability to function efficiently at a wide range of temperatures. Combined molecular modeling and experimental kinetic studies have been carried out for 10S-(+)-trans-anti-[BP]-N2-dG ((+)-ta-[BP]G), a lesion derived from the covalent reaction of a benzo[a]pyrene metabolite with guanine in DNA, at 55 degrees C and results compared with an earlier study at 37 degrees C (Perlow-Poehnelt, R. A., Likhterov, I., Scicchitano, D. A., Geacintov, N. E., and Broyde, S. (2004) J. Biol. Chem. 279, 36951-36961). The experimental results show that there is more overall nucleotide insertion opposite (+)-ta-[BP]G due to particularly enhanced mismatch incorporation at 55 degrees C compared with 37 degrees C. The molecular dynamics simulations suggest that mismatched nucleotide insertion opposite (+)-ta-[BP]G is increased at 55 degrees C compared with 37 degrees C because the higher temperature shifts the preference of the damaged base from the anti to the syn conformation, with the carcinogen on the more open major groove side. The mismatched dNTP structures are less distorted when the damaged base is syn than when it is anti, at the higher temperature. However, with the normal partner dCTP, the anti conformation with close to Watson-Crick alignment remains more favorable. The molecular dynamics simulations are consistent with the kcat values for nucleotide incorporation opposite the lesion studied, providing structural interpretation of the experimental observations. The observed temperature effect suggests that conformational flexibility plays a role in nucleotide incorporation and bypass fidelity opposite (+)-ta-[BP]G by Dpo4.

Nucleotide insertion opposite a cyclobutane pyrimidine dimer analogue caused from photoligation by a replicative DNA polymerase

We previously reported an efficient and reversible template-directed photoligation using 5-carboxyvinyl-2'-deoxyuridine ((CV)U)-containing ODN at the 5'-terminal. This method forms d(T-(CV)U) as a cyclobutane pyrimidine dimer (CPD) analogue between 3'-terminal thymidine and 5'-terminal (CV)U of two oligodeoxynucleotides (ODNs). In this study, we performed PCR using a DNA template containing d(T-(CV)U). And then, we disclosed that two adenines were incorporated opposite the d(T-(CV)U).

Polymerization Past the N2-Isopropylguanine and the N6-Isopropyladenine DNA Lesions with the Translesion Synthesis DNA Polymerases η and ι and the Replicative DNA Polymerase α

The effects of N2-isopropylGua and N6-isopropylAde adducts in template DNA on polymerization by the human replicative DNA polymerase alpha (B-family) and the translesion synthesis DNA polymerases eta and iota (Y-family) were investigated. A direct comparison between the accuracies of DNA synthesis using catalytic fragments of the human DNA polymerases eta and iota is reported. We show that the N2-isopropylGua adduct is a powerful block to polymerization by DNA polymerase alpha. In contrast, the DNA polymerases eta and iota synthesize DNA past the N2-isopropylGua lesion with efficiencies and accuracies opposite the lesion comparable to the unadducted Gua. All three DNA polymerases bypass the N6-isopropylAde adduct with only modest effects on efficiencies and accuracy. These results illustrate the dramatically different consequences to polymerization conferred by the position of the isopropyl adduct when catalyzed by DNA polymerase alpha and the lack of this effect on polymerization by the translesion synthesis DNA polymerases eta and iota. A steady-state kinetic analysis of nucleotide insertion opposite the N2-isopropylGua and the N6-isopropylAde by the DNA polymerases eta and iota was performed to measure the accuracy of DNA synthesis at these lesions. This analysis showed that the DNA polymerases eta and iota preferably insert the correct nucleotide Cyt opposite the N2-isopropylGua lesion and the correct nucleotide Thy opposite the N6-isopropylAde with levels of accuracy similar to those detected opposite the unadducted nucleotides, thus, demonstrating minimal blocking and mutagenic potential by these lesions to the translesion synthesis polymerases. Similarly, a kinetic analysis of polymerization opposite the N6-isopropylAde by the DNA polymerase alpha showed comparable levels of insertion accuracy relative to the unadducted Ade. These results suggest that positioning of the isopropyl adduct on the purine ring to locate this group into the minor groove of the DNA is an important determinant to effect blocked replication by a replicative (B-family) polymerase, but not to affect replication by a translesion synthesis (Y-family) polymerase.

Nucleotide insertion opposite a cis-syn thymine dimer by a replicative DNA polymerase from bacteriophage T7.

Ultraviolet-induced DNA damage poses a lethal block to replication. To understand the structural basis for this, we determined crystal structures of a replicative DNA polymerase from bacteriophage T7 in complex with nucleotide substrates and a DNA template containing a cis-syn cyclobutane pyrimidine dimer (CPD). When the 3' thymine is the templating base, the CPD is rotated out of the polymerase active site and the fingers subdomain adopts an open orientation. When the 5' thymine is the templating base, the CPD lies within the polymerase active site where it base-pairs with the incoming nucleotide and the 3' base of the primer, while the fingers are in a closed conformation. These structures reveal the basis for the strong block of DNA replication that is caused by this photolesion.

Translesion synthesis of acetylaminofluorene-dG adducts by DNA polymerase zeta is stimulated by yeast Rev1 protein.

Translesion synthesis is an important mechanism in response to unrepaired DNA lesions during replication. The DNA polymerase zeta (Polzeta) mutagenesis pathway is a major error-prone translesion synthesis mechanism requiring Polzeta and Rev1. In addition to its dCMP transferase, a non-catalytic function of Rev1 is suspected in cellular response to certain types of DNA lesions. However, it is not well understood about the non-catalytic function of Rev1 in translesion synthesis. We have analyzed the role of Rev1 in translesion synthesis of an acetylaminofluorene (AAF)-dG DNA adduct. Purified yeast Rev1 was essentially unresponsive to a template AAF-dG DNA adduct, in contrast to its efficient C insertion opposite a template 1,N6-ethenoadenine adduct. Purified yeast Polzeta was very inefficient in the bypass of the AAF-dG adduct. Combining Rev1 and Polzeta, however, led to a synergistic effect on translesion synthesis. Rev1 protein enhanced Polzeta-catalyzed nucleotide insertion opposite the AAF-dG adduct and strongly stimulated Polzeta-catalyzed extension from opposite the lesion. Rev1 also stimulated the deficient synthesis by Polzeta at the very end of undamaged DNA templates. Deleting the C-terminal 205 aa of Rev1 did not affect its dCMP transferase activity, but abolished its stimulatory activity on Polzeta-catalyzed extension from opposite the AAF-dG adduct. These results suggest that translesion synthesis of AAF-dG adducts by Polzeta is stimulated by Rev1 protein in yeast. Consistent with the in vitro results, both Polzeta and Rev1 were found to be equally important for error-prone translesion synthesis across from AAF-dG DNA adducts in yeast cells.

The N2-ethylguanine and the O6-ethyl- and O6-methylguanine lesions in DNA: contrasting responses from the "bypass" DNA polymerase eta and the replicative DNA polymerase alpha.

The effects of N(2)-ethylGua, O(6)-ethylGua, and O(6)-methylGua adducts in template DNA on polymerization by mammalian DNA polymerases alpha and eta have been investigated. The N(2)-ethylGua adduct blocks polymerization by the replicative DNA polymerase alpha to a much greater extent than does the O(6)-ethyl- or the O(6)-methylGua adducts. The DNA polymerase eta efficiently and accurately bypasses the N(2)-ethylGua lesion but like DNA polymerase alpha is similarly blocked by the O(6)-ethyl- or the O(6)-methylGua adducts. A steady state kinetic analysis of nucleotide insertion opposite the N(2)-ethylGua and the O(6)-ethylGua adducts by the DNA polymerases alpha and eta and extension from 3'-termini positioned opposite these adducts was performed to measure the efficiency and the accuracy of DNA synthesis past these lesions. This analysis showed that insertion of Cyt opposite the N(2)-ethylGua adduct by DNA polymerase alpha is approximately 10(4)-fold less efficient than insertion of Cyt opposite an unadducted Gua residue at the same position. Extension from the N(2)-ethylGua:Cyt 3'-terminus by DNA polymerase alpha is approximately 10(3)-fold less efficient than extension from a Cyt opposite the unadducted Gua. Insertion of Cyt opposite the N(2)-ethylGua lesion by the DNA polymerase eta is about 370-fold more efficient than by the DNA polymerase alpha, and extension from the N(2)-ethylGua:Cyt 3'-terminus by the DNA polymerase eta is about 3-fold more efficient than by the DNA polymerase alpha. Furthermore, the DNA polymerase eta preferably inserts the correct nucleotide Cyt opposite the N(2)-ethylGua lesion with nearly the same level of accuracy as opposite an unadducted Gua, thus minimizing the mutagentic potential of this lesion. This result contrasts with the relatively high misincorporation efficiency of Thy opposite the O(6)-ethylGua adduct by the DNA polymerases alpha and eta. In reactions containing both DNA polymerases alpha and eta, synthesis past the N(2)-ethylGua adduct is detected to permit completed replication of the adducted oligonucleotide template. These results suggest that accurate replication past the N(2)-ethylGua adduct might be facilitated in cells by pausing of replication catalyzed by DNA polymerase alpha and lesion bypass catalyzed by DNA polymerase eta.

Trans-Lesion Synthesis Past Bulky Benzo[a]pyrene Diol Epoxide N2-dG and N6-dA Lesions Catalyzed by DNA Bypass Polymerases

The effectiveness of in vitro primer elongation reactions catalyzed by human bypass DNA polymerases kappa (hDinB1), pol eta (hRad30A), pol iota (hRad30B), and yeast pol zeta (Rev3 and Rev7) in site-specifically modified template oligonucleotide strands were studied in vitro. The templates contained single bulky lesions derived from the trans-addition of the mutagenic (+)- or (-)-enantiomers of r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (a metabolite of the environmental carcinogen benzo[a]pyrene), to the exocyclic amino groups of guanine or adenine in oligonucleotide templates 33, or more, bases long. In "running start" primer extension reactions, pol kappa effectively bypassed both the stereoisomeric (+)- and (-)-trans-guanine adducts but not the analogous adenine adducts. In sharp contrast, pol eta, which exhibits considerable sequence homology with pol kappa (both belong to the group of Y family polymerases), is partially blocked by the guanine adducts and the (-)-trans-adenine adduct, although the stereoisomeric (+)-trans-adenine adduct is more successfully bypassed. Neither pol iota nor pol zeta, either alone or in combination, were effective in trans-lesion synthesis past the same adducts. In all cases, the fidelity of insertion is dependent on adduct stereochemistry and structure. Generally, error-free nucleotide insertion opposite the lesions tends to depend more on adduct stereochemistry than error-prone insertion. None of the polymerases tested are a universal bypass polymerase for the stereoisomeric bulky polycyclic aromatic hydrocarbon-DNA adducts derived from anti-BPDE.

Miscoding potential of the N2-ethyl-2'-deoxyguanosine DNA adduct by the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I.

Acetaldehyde, a major metabolite of ethanol, reacts with dG residues in DNA, resulting in the formation of the N(2)-ethyl-2'-deoxyguanosine (N(2)-Et-dG) adduct. This adduct has been detected in lymphocyte DNA of alcohol abusers. To explore the miscoding property of the N(2)-Et-dG DNA adduct, phosphoramidite chemical synthesis was used to prepare site-specifically modified oligodeoxynucleotides containing a single N(2)-Et-dG. These N(2)-Et-dG-modified oligodeoxynucleotides were used as templates for primer extension reactions catalyzed by the 3' --> 5' exonuclease-free (exo(-)) Klenow fragment of Escherichia coli DNA polymerase I. The primer extension was retarded one base prior to the N(2)-Et-dG lesion and opposite the lesion; however, when the enzyme was incubated for a longer time or with increased amounts of this enzyme, full extension occurred. Quantitative analysis of the fully extended products showed the preferential incorporation of dGMP and dCMP opposite the N(2)-Et-dG lesion, accompanied by a small amounts of dAMP and dTMP incorporation and one- and two-base deletions. Steady-state kinetic studies were also performed to determine the frequency of nucleotide insertion opposite the N(2)-Et-dG lesion and chain extension from the 3' terminus from the dN.N(2)-Et-dG (N is C, A, G, or T) pairs. These results indicate that the N(2)-Et-dG DNA adduct may generate G --> C transversions in living cells. Such a mutational spectrum has not been detected with other methylated dG adducts, including 8-methyl-2'-deoxyguanosine, O(6)-methyl-2'-deoxyguanosine, and N(2)-methyl-2'-deoxyguanosine. In addition, N(2)-ethyl-2'-deoxyguanosine triphosphate (N(2)-Et-dGTP) was efficiently incorporated opposite a template dC during DNA synthesis catalyzed by the exo(-) Klenow fragment. The utilization of N(2)-Et-dGTP was also determined by steady-state kinetic studies. N(2)-Et-dG DNA adducts are also formed by the incorporation of N(2)-Et-dGTP into DNA and may cause mutations, leading to the development of alcohol- and acetaldehyde-induced human cancers.

Kinetics of DNA polymerase I (Klenow fragment exo-) activity on damaged DNA templates: effect of proximal and distal template damage on DNA synthesis.

Mutagenic DNA adducts have been analyzed with respect to the rate of nucleotide insertion opposite the modified base, extension from that "mispair", and nucleotide insertion preference. To complement and extend these studies we have investigated the long-range effects of DNA adducts on DNA polymerase activity. To address this question, primer extension reactions were performed using DNA polymerase I, Klenow fragment exo-. Templates containing 7,8-dihydro-8-oxoguanine, dG-C8-aminofluorene, dG-C8-(acetylamino)fluorene, and the model abasic site, tetrahydrofuran, were used for these studies, and the steady-state kinetics of correct nucleotide insertion were determined at positions (-2), (-1), (+1), (+2), (+3), and (+5) with respect to the template lesion. The kinetics of primer extension by Klenow fragment exo- at template positions 3' to the lesion showed only a small inhibitory effect, <3-fold, even for the strongly blocking lesion, dG-C8-(acetylamino)fluorene, indicating that Klenow fragment exo- activity is not greatly affected by lesions in the single-stranded portion of the template-primer. In contrast, a dramatic decrease in the frequency of primer extension was observed at template sites 5' to the site of adduction. Inhibition of polymerase activity decreased as the distance from the lesion increased; however, a relatively large effect was seen at the (+2) and (+3) positions for dG-C8-(acetylamino)fluorene and tetrahydrofuran. For these blocking lesions, the effect on extension 5 bases from the lesion was greatly reduced. We conclude from these studies that DNA damage at positions remote from the site of the lesion affects DNA polymerase function.