Role for DNA polymerase kappa in the processing of N2-N2-guanine interstrand cross-links

Although there exists compelling genetic evidence for a homologous recombination-independent pathway for repair of interstrand cross-links (ICLs) involving translesion synthesis (TLS), biochemical support for this model is lacking. To identify DNA polymerases that may function in TLS past ICLs, oligodeoxynucleotides were synthesized containing site-specific ICLs in which the linkage was between N(2)-guanines, similar to cross-links formed by mitomycin C and enals. Here, data are presented that mammalian cell replication of DNAs containing these lesions was approximately 97% accurate. Using a series of oligodeoxynucleotides that mimic potential intermediates in ICL repair, we demonstrate that human polymerase (pol) kappa not only catalyzed accurate incorporation opposite the cross-linked guanine but also replicated beyond the lesion, thus providing the first biochemical evidence for TLS past an ICL. The efficiency of TLS was greatly enhanced by truncation of both the 5 ' and 3 ' ends of the nontemplating strand. Further analyses showed that although yeast Rev1 could incorporate a dCTP opposite the cross-linked guanine, no evidence was found for TLS by pol zeta or a pol zeta/Rev1 combination. Because pol kappa was able to bypass these ICLs, biological evidence for a role for pol kappa in tolerating the N(2)-N(2)-guanine ICLs was sought; both cell survival and chromosomal stability were adversely affected in pol kappa-depleted cells following mitomycin C exposure. Thus, biochemical data and cellular studies both suggest a role for pol kappa in the processing of N(2)-N(2)-guanine ICLs.     

Error-prone translesion synthesis by human DNA polymerase eta on DNA-containing deoxyadenosine adducts of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene

When human DNA polymerase eta (pol eta) encounters N6-deoxyadenosine adducts formed by trans epoxide ring opening of the 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaP DE) isomer with (+)-7R,8S,9S,10R configuration ((+)-BaP DE-2), misincorporation of A or G and incorporation of the correct T are equally likely to occur. On the other hand, the enzyme exhibits a 3-fold preference for correct T incorporation opposite adducts formed by trans ring opening of the (-)-(7S,8R,9R,10S)-DE-2 enantiomer. Adducts at dA formed by cis ring opening of these two BaP DE-2 isomers exhibit a 2-3-fold preference for A over T incorporation, with G intermediate between the two. Extension one nucleotide beyond these adducts is generally weaker than incorporation across from them, but among mismatches the (adducted A*) x A mispair is the most favored for extension. Because mutations can only occur if mispairs are extended, this observation is consistent with the occurrence of A x T to T x A transversions as common mutations in animal cells treated with BaP DE-2 isomers. Adducts with S absolute configuration at the point of attachment of the hydrocarbon to the base inhibit incorporation and extension by pol eta to a lesser extent than their R counterparts. Template-primers containing each of the four isomeric dA adducts derived from BaP DE-2 and two adducts derived from 9,10-epoxy-7,8,9,10-tetrahydrobenzo-[a]pyrene in which the 7- and 8-hydroxyl groups of the DEs are replaced with hydrogens exhibit reduced electrophoretic mobilities relative to the unadducted oligonucleotides. This effect is largely independent of DNA sequence. Decreased mobility correlates with an increased rate of incorporation by pol eta, suggesting a systematic relationship between the overall DNA structure and efficiency of the enzyme.     

Oxygen free radical damage to DNA. Translesion synthesis by human DNA polymerase eta and resistance to exonuclease action at cyclopurine deoxynucleoside residues.

Cyclopurine deoxynucleosides are common DNA lesions generated by exposure to reactive oxygen species under hypoxic conditions. The S and R diastereoisomers of cyclodeoxyadenosine on DNA were investigated separately for their ability to block 3' to 5' exonucleases. The mammalian DNA-editing enzyme DNase III (TREX1) was blocked by both diastereoisomers, whereas only the S diastereoisomer was highly efficient in preventing digestion by the exonuclease function of T4 DNA polymerase. Digestion in both cases was frequently blocked one residue before the modified base. Oligodeoxyribonucleotides containing a cyclodeoxyadenosine residue were further employed as templates for synthesis by human DNA polymerase eta (pol eta). pol eta could catalyze translesion synthesis on the R diastereoisomer of cyclodeoxyadenosine. On the S diastereoisomer, pol eta could catalyze the incorporation of one nucleotide opposite the lesion but could not continue elongation. Although pol eta preferentially incorporated dAMP opposite the R diastereoisomer, elongation continued only when dTMP was incorporated, suggesting bypass of this lesion by pol eta with reasonable fidelity. With the S diastereoisomer, pol eta mainly incorporated dAMP or dTMP opposite the lesion but could not elongate even after incorporating a correct nucleotide. These data suggest that the S diastereoisomer may be a more cytotoxic DNA lesion than the R diastereoisomer.     

Kinetic evidence for inefficient and error-prone bypass across bulky N2-guanine DNA adducts by human DNA polymerase iota

DNA polymerase (pol) iota has been proposed to be involved in translesion synthesis past minor groove DNA adducts via Hoogsteen base pairing. The N2 position of G, located in minor groove side of duplex DNA, is a major site for DNA modification by various carcinogens. Oligonucleotides with varying adduct size at G N2 were analyzed for bypass ability and fidelity with human pol iota. Pol iota effectively bypassed N2-methyl (Me)G and N2-ethyl(Et)G, partially bypassed N2-isobutyl(Ib)G and N2-benzylG, and was blocked at N2-CH2(2-naphthyl)G (N2-NaphG), N2-CH2(9-anthracenyl)G (N2-AnthG), and N2-CH2(6-benzo[a]pyrenyl)G. Steady-state kinetic analysis showed decreases of kcat/Km for dCTP insertion opposite N2-G adducts according to size, with a maximal decrease opposite N2-AnthG (61-fold). dTTP misinsertion frequency opposite template G was increased 3-11-fold opposite adducts (highest with N2-NaphG), indicating the additive effect of bulk (or possibly hydrophobicity) on T misincorporation. N2-IbG, N2-NaphG, and N2-AnthG also decreased the pre-steady-state kinetic burst rate compared with unmodified G. High kinetic thio effects (S(p)-2'-deoxycytidine 5'-O-(1-thiotriphosphate)) opposite N2-EtG and N2-AnthG (but not G) suggest that the chemistry step is largely interfered with by adducts. Severe inhibition of polymerization opposite N2,N2-diMeG compared with N2-EtG by pol eta but not by pol iota is consistent with Hoogsteen base pairing by pol iota. Thus, polymerization by pol iota is severely inhibited by a bulky group at G N2 despite an advantageous mode of Hoogsteen base pairing; pol iota may play a limited role in translesion synthesis on bulky N2-G adducts in cells.     

Biochemical basis of genotoxicity of heterocyclic arylamine food mutagens: Human DNA polymerase eta selectively produces a two-base deletion in copying the N2-guanyl adduct of 2-amino-3-methylimidazo[4,5-f]quinoline but not the C8 adduct at the NarI G3 site

Heterocyclic arylamines are highly mutagenic and cause tumors in animal models. The mutagenicity is attributed to the C8- and N2-G adducts, the latter of which accumulates due to slower repair. The C8- and N 2-G adducts derived from 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) were placed at the G1 and G3 sites of the NarI sequence, in which the G3 site is an established hot spot for frameshift mutation with the model arylamine derivative 2-acetylaminofluorene but G1 is not. Human DNA polymerase (pol) eta extended primers beyond template G-IQ adducts better than did pol kappa and much better than pol iota or delta. In 1-base incorporation studies, pol eta inserted C and A, pol iota inserted T, and pol kappa inserted G. Steady-state kinetic parameters were measured for these dNTPs opposite the C8- and N 2-IQ adducts at both sites, being most favorable for pol eta. Mass spectrometry of pol eta extension products revealed a single major product in each of four cases; with the G1 and G3 C8-IQ adducts, incorporation was largely error-free. With the G3 N 2-IQ adduct, a -2 deletion occurred at the site of the adduct. With the G1 N 2-IQ adduct, the product was error-free at the site opposite the base and then stalled. Thus, the pol eta products yielded frame-shifts with the N 2 but not the C8 IQ adducts. We show a role for pol eta and the complexity of different chemical adducts of IQ, DNA position, and DNA polymerases.     

Interaction with PCNA is essential for yeast DNA polymerase eta function.

In both yeast and humans, DNA polymerase (Pol) eta functions in error-free replication of ultraviolet-damaged DNA, and Poleta promotes replication through many other DNA lesions as well. Here, we present evidence for the physical and functional interaction of yeast Poleta with proliferating cell nuclear antigen (PCNA) and show that the interaction with PCNA is essential for the in vivo function of Poleta. Poleta is highly inefficient at inserting a nucleotide opposite an abasic site, but interaction with PCNA greatly stimulates its ability for nucleotide incorporation opposite this lesion. Thus, in addition to having a pivotal role in the targeting of Poleta to the replication machinery stalled at DNA lesions, interaction with PCNA would promote the bypass of certain DNA lesions.     

Translesion synthesis across bulky N2-alkyl guanine DNA adducts by human DNA polymerase kappa

DNA polymerase (pol) kappa is one of the so-called translesion polymerases involved in replication past DNA lesions. Bypass events have been studied with a number of chemical modifications with human pol kappa, and the conclusion has been presented, based on limited quantitative data, that the enzyme is ineffective at incorporating opposite DNA damage but proficient at extending beyond bases paired with the damage. Purified recombinant full-length human pol kappa was studied with a series of eight N(2)-guanyl adducts (in oligonucleotides) ranging in size from methyl- to -CH(2)(6-benzo[a]pyrenyl) (BP). Steady-state kinetic parameters (catalytic specificity, k(cat)/K(m)) were similar for insertion of dCTP opposite the lesions and for extension beyond the N(2)-adduct G:C pairs. Mispairing of dGTP and dTTP was similar and occurred with k(cat)/K(m) values approximately 10(-3) less than for dCTP with all adducts; a similar differential was found for extension beyond a paired adduct. Pre-steady-state kinetic analysis showed moderately rapid burst kinetics for dCTP incorporations, even opposite the bulky methyl(9-anthracenyl)- and BPG adducts (k(p) 5.9-10.3 s(-1)). The rapid bursts were abolished opposite BPG when alpha-thio-dCTP was used instead of dCTP, implying rate-limiting phosphodiester bond formation. Comparisons are made with similar studies done with human pols eta and iota; pol kappa is the most resistant to N(2)-bulk and the most quantitatively efficient of these in catalyzing dCTP incorporation opposite bulky guanine N(2)-adducts, particularly the largest (N(2)-BPG).     

Preferential misincorporation of purine nucleotides by human DNA polymerase eta opposite benzo[a]pyrene 7,8-diol 9,10-epoxide deoxyguanosine adducts

Human DNA polymerase eta was used to copy four stereoisomeric deoxyguanosine (dG) adducts derived from benzo[a]pyrene 7,8-diol 9,10-epoxide (diastereomer with the 7-hydroxyl group and epoxide oxygen trans (BaP DE-2)). The adducts, formed by either cis or trans epoxide ring opening of each enantiomer of BaP DE-2 by N(2) of dG, were placed at the fourth nucleotide from the 5'-end in two 16-mer sequence contexts, 5' approximately CG*A approximately and 5' approximately GG*T. poleta was remarkably error prone at all four diol epoxide adducts, preferring to misincorporate G and A at frequencies 3- to more than 50-fold greater than the frequencies for T or the correct C, although the highest rates were 60-fold below the rate of incorporation of C opposite a non-adducted G. Anti to syn rotation of the adducted base, consistent with previous NMR data for a BaP DE-2 dG adduct placed just beyond a primer terminus, provides a rationale for preferring purine misincorporation. Extension of purine misincorporations occurred preferentially, but extension beyond the adduct site was weak with V(max)/K(m) values generally 10-fold less than for misincorporation. Mostly A was incorporated opposite (+)-BaP DE-2 dG adducts, which correlates with published observations that G --> T is the most common type of mutation that (+)-BaP DE-2 induces in mammalian cells.     

Mechanisms of accurate translesion synthesis by human DNA polymerase eta

The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta (pol eta), which is involved in the replication of damaged DNA. Pol eta catalyzes efficient and accurate translesion synthesis past cis-syn cyclobutane di-thymine lesions. Here we show that human pol eta can catalyze translesion synthesis past an abasic (AP) site analog, N-2-acetylaminofluorene (AAF)-modified guanine, and a cisplatin-induced intrastrand cross-link between two guanines. Pol eta preferentially incorporated dAMP and dGMP opposite AP, and dCMP opposite AAF-G and cisplatin-GG, but other nucleotides were also incorporated opposite these lesions. However, after incorporating an incorrect nucleotide opposite a lesion, pol eta could not continue chain elongation. In contrast, after incorporating the correct nucleotide opposite a lesion, pol eta could continue chain elongation, whereas pol alpha could not. Thus, the fidelity of translesion synthesis by human pol eta relies not only on the ability of this enzyme to incorporate the correct nucleotide opposite a lesion, but also on its ability to elongate only DNA chains that have a correctly incorporated nucleotide opposite a lesion.     

Mechanism of efficient and accurate nucleotide incorporation opposite 7,8-dihydro-8-oxoguanine by Saccharomyces cerevisiae DNA polymerase eta

Most DNA polymerases incorporate nucleotides opposite template 7,8-dihydro-8-oxoguanine (8-oxoG) lesions with reduced efficiency and accuracy. DNA polymerase (Pol) eta, which catalyzes the error-free replication of template thymine-thymine (TT) dimers, has the unique ability to accurately and efficiently incorporate nucleotides opposite 8-oxoG templates. Here we have used pre-steady-state kinetics to examine the mechanisms of correct and incorrect nucleotide incorporation opposite G and 8-oxoG by Saccharomyces cerevisiae Pol eta. We found that Pol eta binds the incoming correct dCTP opposite both G and 8-oxoG with similar affinities, and it incorporates the correct nucleotide bound opposite both G and 8-oxoG with similar rates. While Pol eta incorporates an incorrect A opposite 8-oxoG with lower efficiency than it incorporates a correct C, it does incorporate A more efficiently opposite 8-oxoG than opposite G. This is mainly due to greater binding affinity for the incorrect incoming dATP opposite 8-oxoG. Overall, these results show that Pol eta replicates through 8-oxoG without any barriers introduced by the presence of the lesion.     

Butadiene-induced intrastrand DNA cross-links: a possible role in deletion mutagenesis

To initiate studies designed to identify the mutagenic spectrum associated with butadiene diepoxide-induced N(2)-N(2) guanine intrastrand cross-links, site specifically adducted oligodeoxynucleotides were synthesized in which the adducted bases were centrally located within the context of the human ras 12 codon. The two stereospecifically modified DNAs and the corresponding unmodified DNA were ligated into a single-stranded M13mp7L2 vector and transfected into Escherichia coli. Both stereoisomeric forms (R, R and S,S) of the DNA cross-links resulted in very severely decreased plaque-forming ability, along with an increased mutagenic frequency for both single base substitutions and deletions compared with unadducted DNAs, with the S,S stereoisomer being the most mutagenic. Consistent with decreased plaque formation, in vitro replication of DNA templates containing the cross-links by the three major E. coli polymerases revealed replication blockage by both stereoisomeric forms of the cross-links. The same DNAs that were used for replication studies were also assembled into duplex DNAs and tested as substrates for the initiation of nucleotide excision repair by the E. coli UvrABC complex. UvrABC incised linear substrates containing these intrastrand cross-links with low efficiency, suggesting that these lesions may be inefficiently repaired by the nucleotide excision repair system.     

Mutational specificity of gamma-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 eta

Comparative mutagenesis of gamma- 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 approximately 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 eta (hpol eta) 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 eta can bypass the cross-link incorporating the correct dNMP opposite each cross-linked base. For G[8,5-Me]T, nucleotide incorporation by hpol eta was significantly different from that by yeast pol eta in that the latter was more error-prone opposite the cross-linked Gua. The incorporation of the correct nucleotide, dAMP, by hpol eta 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 eta was not consistent with the observed cellular mutations. Nevertheless, at and near the lesion, hpol eta 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 eta may play a role in correct bypass of the cross-links.     

Fidelity of nucleotide insertion at 8-oxo-7,8-dihydroguanine by mammalian DNA polymerase delta. Steady-state and pre-steady-state kinetic analysis

Nucleotide insertion opposite 8-oxo-7,8-dihydroguanine (8-oxoG) by fetal calf thymus DNA polymerase delta (pol delta) was examined by steady-state and pre-steady-state rapid quench kinetic analyses. In steady-state reactions with the accessory protein proliferating cell nuclear antigen (PCNA), pol delta preferred to incorporate dCTP opposite 8-oxoG with an efficiency of incorporation an order of magnitude lower than incorporation into unmodified DNA (mainly due to an increased K(m)). Pre-steady-state kinetic analysis of incorporation opposite 8-oxoG showed biphasic kinetics for incorporation of either dCTP or dATP, with rates similar to dCTP incorporation opposite G, large phosphorothioate effects (>100), and oligonucleotide dissociation apparently rate-limiting in the steady-state. Although pol delta preferred to incorporate dCTP (14% misincorporation of dATP) the extension past the A:8-oxoG mispair predominated. The presence of PCNA was found to be a more essential factor for nucleotide incorporation opposite 8-oxoG adducts than unmodified DNA, increased pre-steady-state rates of nucleotide incorporation by >2 orders of magnitude, and was essential for nucleotide extension beyond 8-oxoG. pol delta replication fidelity at 8-oxoG depends upon contributions from K(m), K(d)(dNTP), and rates of phosphodiester bond formation, and PCNA is an important accessory protein for incorporation and extension at 8-oxoG adducts.     

DNA polymerase action blocked by adenine adducts induced by 5-hydroxymethylchrysene sulfate.

Modification of M13mp10 single-stranded DNA with 5-hydroxymethylchrysene (5HCR) sulfate, the ultimate carcinogenic metabolite of 5-methylchrysene, resulted in formation of N6[(chrysen-5-yl)methyl]adenine and N2[(chrysen-5-yl)methyl]-guanine at the ratio of 2.7:1. Measurement of DNA synthesis using this modified template and E.coli DNA polymerase I (Klenow fragment) demonstrated that increasing levels of adducts caused a progressive decline in replication. Analysis of reaction products on DNA-sequence gels revealed DNA elongation to be arrested exclusively at adenine adducts in -AAAGGA- and -AACA- sequences.     

Miscoding properties of 1,N6-ethanoadenine, a DNA adduct derived from reaction with the antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea

1,N(6)-Ethanoadenine (EA) is an exocyclic adduct formed from DNA reaction with the antitumor agent, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). To understand the role of this adduct in the mechanism of mutagenicity or carcinogenicity by BCNU, an oligonucleotide with a site-specific EA was synthesized using phosphoramidite chemistry. We now report the in vitro miscoding properties of EA in translesion DNA synthesis catalyzed by mammalian DNA polymerases (pols) alpha, beta, eta and iota. These data were also compared with those obtained for the structurally related exocyclic adduct, 1,N(6)-ethenoadenine (epsilonA). Using a primer extension assay, both pols alpha and beta were primarily blocked by EA or epsilonA with very minor extension. Pol eta, a member of the Y family of polymerases, was capable of catalyzing a significant amount of bypass across both adducts. Pol eta incorporated all four nucleotides opposite EA and epsilonA, but with differential preferences and mainly in an error-prone manner. Human pol iota, a paralog of human pol eta, was blocked by both adducts with a very small amount of synthesis past epsilonA. It incorporated C and, to a much lesser extent, T, opposite either adduct. In addition, the presence of an A adduct, e.g. epsilonA, could affect the specificity of pol iota toward the template T immediately 3' to the adduct. In conclusion, the four polymerases assayed on templates containing an EA or epsilonA showed differential bypass capacity and nucleotide incorporation specificity, with the two adducts not completely identical in influencing these properties. Although there was a measurable extent of error-free nucleotide incorporation, all these polymerases primarily misincorporated opposite EA, indicating that the adduct, similar to epsilonA, is a miscoding lesion.     

Two-step error-prone bypass of the (+)- and (-)-trans-anti-BPDE-N2-dG adducts by human DNA polymerases eta and kappa

Benzo[a]pyrene is a polycyclic aromatic hydrocarbon (PAH) associated with potent carcinogenic activity. Mutagenesis induced by benzo[a]pyrene DNA adducts is believed to involve error-prone translesion synthesis opposite the lesion. However, the DNA polymerase involved in this process has not been clearly defined in eukaryotes. Here, we provide biochemical evidence suggesting a role for DNA polymerase eta (Poleta) in mutagenesis induced by benzo[a]pyrene DNA adducts in cells. Purified human Poleta predominantly inserted an A opposite a template (+)- and (-)-trans-anti-BPDE-N2-dG, two important DNA adducts of benzo[a]pyrene. Both lesions also dramatically elevated G and T mis-insertion error rates of human Poleta. Error-prone nucleotide insertion by human Poleta was more efficient opposite the (+)-trans-anti-BPDE-N2-dG adduct than opposite the (-)-trans-anti-BPDE-N2-dG. However, translesion synthesis by human Poleta largely stopped opposite the lesion and at one nucleotide downstream of the lesion (+1 extension). The limited extension synthesis of human Poleta from opposite the lesion was strongly affected by the stereochemistry of the trans-anti-BPDE-N2-dG adducts, the nucleotide opposite the lesion, and the sequence context 5' to the lesion. By combining the nucleotide insertion activity of human Poleta and the extension synthesis activity of human Polkappa, effective error-prone lesion bypass was achieved in vitro in response to the (+)- and (-)-trans-anti-BPDE-N2-dG DNA adducts.     

Kinetic analysis of translesion synthesis opposite bulky N2- and O6-alkylguanine DNA adducts by human DNA polymerase REV1

REV1, a Y family DNA polymerase (pol), is involved in replicative bypass past DNA lesions, so-called translesion DNA synthesis. In addition to a structural role as a scaffold protein, REV1 has been proposed to play a catalytic role as a dCTP transferase in translesion DNA synthesis past abasic and guanine lesions in eukaryotes. To better understand the catalytic function of REV1 in guanine lesion bypass, purified recombinant human REV1 was studied with two series of guanine lesions, N(2)-alkylG adducts (in oligonucleotides) ranging in size from methyl (Me) to CH(2)(6-benzo[a]pyrenyl) (BP) and O(6)-alkylG adducts ranging from Me to 4-oxo-4-(3-pyridyl)butyl (Pob). REV1 readily produced 1-base incorporation opposite G and all G adducts except for O(6)-PobG, which caused almost complete blockage. Steady-state kinetic parameters (k(cat)/K(m)) were similar for insertion of dCTP opposite G and N(2)-G adducts but were severely reduced opposite the O(6)-G adducts. REV1 showed apparent pre-steady-state burst kinetics for dCTP incorporation only opposite N(2)-BPG and little, if any, opposite G, N(2)-benzyl (Bz)G, or O(6)-BzG. The maximal polymerization rate (k(pol) 0.9 s(-1)) opposite N(2)-BPG was almost the same as opposite G, with only slightly decreased binding affinity to dCTP (2.5-fold). REV1 bound N(2)-BPG-adducted DNA 3-fold more tightly than unmodified G-containing DNA. These results and the lack of an elemental effect ((S(p))-2'-deoxycytidine 5'-O-(1-thiotriphosphate)) suggest that the late steps after product formation (possibly product release) become rate-limiting in catalysis opposite N(2)-BPG. We conclude that human REV1, apparently the slowest Y family polymerase, is kinetically highly tolerant to N(2)-adduct at G but not to O(6)-adducts.     

Replication bypass of interstrand cross-link intermediates by Escherichia coli DNA polymerase IV

Repair of interstrand DNA cross-links (ICLs) in Escherichia coli can occur through a combination of nucleotide excision repair (NER) and homologous recombination. However, an alternative mechanism has been proposed in which repair is initiated by NER followed by translesion DNA synthesis (TLS) and completed through another round of NER. Using site-specifically modified oligodeoxynucleotides that serve as a model for potential repair intermediates following incision by E. coli NER proteins, the ability of E. coli DNA polymerases (pol) II and IV to catalyze TLS past N(2)-N(2)-guanine ICLs was determined. No biochemical evidence was found suggesting that pol II could bypass these lesions. In contrast, pol IV could catalyze TLS when the nucleotides that are 5' to the cross-link were removed. The efficiency of TLS was further increased when the nucleotides 3' to the cross-linked site were also removed. The correct nucleotide, C, was preferentially incorporated opposite the lesion. When E. coli cells were transformed with a vector carrying a site-specific N(2)-N(2)-guanine ICL, the transformation efficiency of a pol II-deficient strain was indistinguishable from that of the wild type. However, the ability to replicate the modified vector DNA was nearly abolished in a pol IV-deficient strain. These data strongly suggest that pol IV is responsible for TLS past N(2)-N(2)-guanine ICLs.     

Replication past a trans-4-hydroxynonenal minor-groove adduct by the sequential action of human DNA polymerases iota and kappa

The X-ray crystal structure of human DNA polymerase iota (Poliota) has shown that it differs from all known Pols in its dependence upon Hoogsteen base pairing for synthesizing DNA. Hoogsteen base pairing provides an elegant mechanism for synthesizing DNA opposite minor-groove adducts that present a severe block to synthesis by replicative DNA polymerases. Germane to this problem, a variety of DNA adducts form at the N2 minor-groove position of guanine. Previously, we have shown that proficient and error-free replication through the gamma-HOPdG (gamma-hydroxy-1,N2-propano-2'-deoxyguanosine) adduct, which is formed from the reaction of acrolein with the N2 of guanine, is mediated by the sequential action of human Poliota and Polkappa, in which Poliota incorporates the nucleotide opposite the lesion site and Polkappa carries out the subsequent extension reaction. To test the general applicability of these observations to other adducts formed at the N2 position of guanine, here we examine the proficiency of human Poliota and Polkappa to synthesize past stereoisomers of trans-4-hydroxy-2-nonenal-deoxyguanosine (HNE-dG). Even though HNE- and acrolein-modified dGs share common structural features, due to their increased size and other structural differences, HNE adducts are potentially more blocking for replication than gamma-HOPdG. We show here that the sequential action of Poliota and Polkappa promotes efficient and error-free synthesis through the HNE-dG adducts, in which Poliota incorporates the nucleotide opposite the lesion site and Polkappa performs the extension reaction.