MutS recognition of exocyclic DNA adducts that are endogenous products of lipid oxidation.

The ability of the methyl-directed mismatch repair system to recognize and repair the exocyclic adducts propanodeoxyguanosine (PdG) and pyrimido[1,2-alpha]purin-10(3H)-one (M(1)G), the major adduct derived from the endogenous mutagen malondialdehyde, has been assessed both in vivo and in vitro. Both adducts were site-specifically incorporated into M13MB102 DNA, and the adducted genomes were electroporated into wild-type or mutS-deficient Escherichia coli strains. A decrease in mutations caused by both adducts was observed in mutS-deficient strains, suggesting that MutS was binding to the adducts and blocking repair by nucleotide excision repair. This hypothesis was supported by the differences in mutation frequency observed when hemimethylated genomes containing PdG on the (-)-strand were electroporated into a uvrA(-) strain. The ability of purified MutS to bind to PdG- or M(1)G-containing 31-mer duplexes in vitro was assessed using both surface plasmon resonance and gel shift assays. MutS bound to M(1)G:T-containing duplexes with similar affinity to a G:T mismatch but less strongly to M(1)G:C- and PdG-containing duplexes. Dissociation from each of the adduct-containing duplexes occurred at a faster rate than from a G:T mismatch. The present results indicate that MutS can bind to exocyclic adducts resulting from endogenous DNA damage and trigger their removal by mismatch repair or protect them from removal by nucleotide excision repair.     

Site-specific frameshift mutagenesis by a propanodeoxyguanosine adduct positioned in the (CpG)4 hot-spot of Salmonella typhimurium hisD3052 carried on an M13 vector.

Malondialdehyde induces frameshift mutations in Salmonella typhimurium strain hisD3052. The ability of propanodeoxyguanosine (PdG), a structural analog of the major malondialdehyde-deoxyguanosine adduct, to induce site-specific frameshift mutations was tested in the (CpG)4 hot-spot of hisD3052 carried on an M13 vector (M13MB102). PdG was introduced at position 6248 of duplex M13MB102 by ligation of the oligonucleotide 5'-CGC(PdG)CGGCATG-3' into a heteroduplex containing an 11-nucleotide gap in the (-)-strand between the SphI and BssHII restriction sites and deoxyuridine in place of thymidine in the (+)-strand. Ligation proceeded with 70% efficiency, and closed circular duplex DNA molecules were isolated in 40% yield. The adducted genome was sensitive to cleavage by SphI but resistant to cleavage by BssHII. Transformation of Escherichia coli strain JM105 with adducted M13MB102 led to 25% reduced survival relative to unadducted M13MB102 and produced frameshift mutations in 2.5% of the progeny phage. All of the mutations were deletions, and 70% occurred by deletion of CpG. Unadducted genomes exhibited a 40-fold lower mutation frequency, and all the mutations were single-base deletions at the sites of ligation of the 11-mer. These results illustrate that PdG, a structural analog of the major malondialdehyde-deoxyguanosine adduct, induces frameshift mutations in M13MB102 and that single-stranded nicks are efficient premutagenic lesions in this recombinant bacteriophage.     

Cell cycle-dependent strand bias for UV-induced mutations in the transcribed strand of excision repair-proficient human fibroblasts but not in repair-deficient cells.

To study the effect of nucleotide excision repair on the spectrum of mutations induced in diploid human fibroblasts by UV light (wavelength, 254 nm), we synchronized repair-proficient cells and irradiated them when the HPRT gene was about to be replicated (early S phase) so that there would be no time for repair in that gene before replication, or in G1 phase 6 h prior to S, and determined the kinds and location of mutations in that gene. As a control, we also compared the spectra of mutations induced in synchronized populations of xeroderma pigmentosum cells (XP12BE cells, which are unable to excise UV-induced DNA damage). Among the 84 mutants sequenced, base substitutions predominated. Of the XP mutants from S or G1 and the repair-proficient mutants from S, approximately 62% were G.C----A.T. In the repair-proficient mutants from G1, 47% were. In mutants from the repair-proficient cells irradiated in S, 71% (10 of 14) of the premutagenic lesions were located in the transcribed strand; with mutants from such cells irradiated in G1, only 20% (3 of 15) were. In contrast, there was no statistically significant difference in the fraction of premutagenic lesions located in the transcribed strand of the XP12BE cells; approximately 75% (24 of 32) of the premutagenic lesions were located in that strand, i.e., 15 of 19 (79%) in the S-phase cells and 9 of 13 (69%) in the G1-phase cells. The switch in strand bias supports preferential nucleotide excision repair of UV-induced damage in the transcribed strand of the HPRT gene.     

Requirements for West Nile virus (-)- and (+)-strand subgenomic RNA synthesis in vitro by the viral RNA-dependent RNA polymerase expressed in Escherichia coli

RNA-dependent RNA polymerases (RdRPs) of the Flaviviridae family catalyze replication of positive (+)- strand viral RNA through synthesis of minus (-)-and progeny (+)-strand RNAs. West Nile virus (WNV), a mosquito-borne member, is a rapidly re-emerging human pathogen in the United States since its first outbreak in 1999. To study the replication of the WNV RNA in vitro, an assay is described here that utilizes the WNV RdRP and subgenomic (-)- and (+)-strand template RNAs containing 5'- and 3'-terminal regions (TR) with the conserved sequence elements. Our results show that both 5'- and 3'-TRs of the (+)-strand RNA template including the wild type cyclization (CYC) motifs are important for RNA synthesis. However, the 3'-TR of the (-)-strand RNA template alone is sufficient for RNA synthesis. Mutational analysis of the CYC motifs revealed that the (+)-strand 5'-CYC motif is critical for (-)-strand RNA synthesis but neither the (-)-strand 5'- nor 3'-CYC motif is important for the (+)-strand RNA synthesis. Moreover, the 5'-cap inhibits the (-)-strand RNA synthesis from the 3' fold-back structure of (+)-strand RNA template without affecting the de novo synthesis of RNA. These results support a model that "cyclization" of the viral RNA play a role for (-)-strand RNA synthesis but not for (+)-strand RNA synthesis.     

Emergence of distinct brome mosaic virus recombinants is determined by the polarity of the inoculum RNA

Despite overwhelming interest in the impact exerted by recombination during evolution of RNA viruses, the relative contribution of the polarity of inoculum templates remains poorly understood. Here, by agroinfiltrating Nicotiana benthamiana leaves, we show that brome mosaic virus (BMV) replicase is competent to initiate positive-strand [(+)-strand] synthesis on an ectopically expressed RNA3 negative strand [(-) strand] and faithfully complete the replication cycle. Consequently, we sought to examine the role of RNA polarity in BMV recombination by expressing a series of replication-defective mutants of BMV RNA3 in (+) or (-) polarity. Temporal analysis of progeny sequences revealed that the genetic makeup of the primary recombinant pool is determined by the polarity of the inoculum template. When the polarity of the inoculum template was (+), the recombinant pool that accumulated during early phases of replication was a mixture of nonhomologous recombinants. These are longer than the inoculum template length, and a nascent 3' untranslated region (UTR) of wild-type (WT) RNA1 or RNA2 was added to the input mutant RNA3 3' UTR due to end-to-end template switching by BMV replicase during (-)-strand synthesis. In contrast, when the polarity of the inoculum was (-), the progeny contained a pool of native-length homologous recombinants generated by template switching of BMV replicase with a nascent UTR from WT RNA1 or RNA2 during (+)-strand synthesis. Repair of a point mutation caused by polymerase error occurred only when the polarity of the inoculum template was (+). These results contribute to the explanation of the functional role of RNA polarity in recombination mediated by copy choice mechanisms.     

Substitution and deletion mutations induced by 2-hydroxyadenine in Escherichia coli: effects of sequence contexts in leading and lagging strands.

To evaluate the mutation frequency and the mutation spectrum of 2-hydroxyadenine (2-OH-Ade), an oxidative DNA lesion, the modified base was site-specifically incorporated into a unique restriction enzyme site (SalI, GTCGA*C or AflII, CTTA*AG where A* represents 2-OH-Ade) in single- and double-stranded vectors. The 2-OH-Ade residues were introduced into (+)- and (-)-strands of the double-stranded vectors and into the (+)-strand of single-stranded vectors. When the vectors were transfected intoEscherichia coli, the modified base showed little to no cytotoxicity. The mutation frequencies of 2-OH-Ade in the SalI and AflII sites were approximately 0.8 and 0.07%, respectively, with double-stranded (+)-vectors. An increase in the mutation frequencies was not observed with single-stranded vectors. When incorporated into the (-)-strand, the mutation frequencies of 2-OH-Ade in the SalI and AflII sites were approximately 0.3 and 0.1%, respectively. The mutations observed most frequently were -1 deletions at both positions, in the case of the (+)-strand. On the other hand, we observed that 2-OH-Ade in the (-)-strand induced A-->G and A-->T substitutions. These results indicate that 2-OH-Ade residues in DNA induce substitution and deletion mutations without blocking replication inE.coli.     

Mutagenic and genotoxic effects of DNA adducts formed by the anticancer drug cis-diamminedichloroplatinum(II).

The toxicity and mutagenicity of three DNA adducts formed by the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP or cisplatin) were investigated in Escherichia coli. The adducts studied were cis-[Pt(NH3)2(d(GpG))] (G*G*), cis-[Pt(NH3)2(d(ApG))] (A*G*) and cis-[Pt(NH3)2(d(GpTpG))] (G*TG*), which collectively represent approximately 95% of the DNA adducts reported to form when the drug damages DNA. Oligonucleotide 24-mers containing each adduct were positioned at a known site within the viral strand of single stranded M13mp7L2 bacteriophage DNA. Following transfection into E. coli DL7 cells, the genomes containing the G*G*, A*G* and G*TG* adducts had survival levels of 5.2 +/- 1.2, 22 +/- 2.6 and 14 +/- 2.5% respectively, compared to unmodified genomes. Upon SOS induction, the survival of genomes containing the G*G* and A*G* adducts increased to 31 +/- 5.4 and 32 +/- 4.9% respectively. Survival of the genome containing the G*TG* adduct did not increase upon SOS induction. In SOS induced cells, the G*G* and A*G* adducts gave rise predominantly to G-->T and A-->T transversions respectively, targeted to the 5' modified base. In addition, A-->G transitions were detected for the A*G* adduct and low levels of tandem mutations at the 5' modified base as well as the adjacent 5' base were also observed for both adducts. The A*G* adduct was more mutagenic than the G*G* adduct, with a mutation frequency of 6% compared to 1.4% for the latter adduct. No cis-[Pt(NH3)2)2+ intrastrand crosslink-specific mutations were observed for the G*TG* adduct.     

Activities of human DNA polymerase kappa in response to the major benzo[a]pyrene DNA adduct: error-free lesion bypass and extension synthesis from opposite the lesion

In cells, the major benzo[a]pyrene DNA adduct is the highly mutagenic (+)-trans-anti-BPDE-N(2)-dG. In eukaryotes, little is known about lesion bypass of this DNA adduct during replication. Here, we show that purified human Polkappa can effectively bypass a template (+)-trans-anti-BPDE-N(2)-dG adduct in an error-free manner. Kinetic parameters indicate that Polkappa bypass of the (-)-trans-anti-BPDE-N(2)-dG adduct was approximately 41-fold more efficient compared to the (+)-trans-anti-BPDE-N(2)-dG adduct. Furthermore, we have found another activity of human Polkappa in response to the (+)- and (-)-trans-anti-BPDE-N(2)-dG adducts: extension synthesis from mispaired primer 3' ends opposite the lesion. In contrast, the two adducts strongly blocked DNA synthesis by the purified human Polbeta and the purified catalytic subunits of yeast Polalpha, Poldelta, and Pol epsilon right before the lesion. Extension by human Polkappa from the primer 3' G opposite the (+)- and (-)-trans-anti-BPDE-N(2)-dG adducts was mediated by a -1 deletion mechanism, probably resulting from re-aligning the primer G to pair with the next template C by Polkappa prior to DNA synthesis. Thus, sequence contexts 5' to the lesion strongly affect the fidelity and mechanism of the Polkappa-catalyzed extension synthesis. These results support a dual-function model of human Polkappa in bypass of BPDE DNA adducts: it may function both as an error-free bypass polymerase alone and an extension synthesis polymerase in combination with another polymerase.     

Effect of sequence context on O(6)-methylguanine repair and replication in vivo.

Understanding the origins of mutational hotspots is complicated by the intertwining of several variables. The selective formation, repair, and replication of a DNA lesion, such as O(6)-methylguanine (m(6)G), can, in principle, be influenced by the surrounding nucleotide environment. A nearest-neighbor analysis was used to address the contribution of sequence context on m(6)G repair by the Escherichia coli methyltransferases Ada or Ogt, and on DNA polymerase infidelity in vivo. Sixteen M13 viral genomes with m(6)G flanked by all permutations of G, A, T, and C were constructed and individually transformed into repair-deficient and repair-proficient isogenic cell strains. The 16 genomes were introduced in duplicate into 5 different cellular backgrounds for a total of 160 independent experiments, for which mutations were scored using a recently developed assay. The Ada methyltransferase demonstrated strong 5' and 3' sequence-specific repair of m(6)G in vivo. The Ada 5' preference decreased in the general order: GXN > CXN > TXN > AXN (X = m(6)G, N = any base), while the Ada 3' preference decreased in the order: NX(T/C) > NX(G/A), with mutation frequencies (MFs) ranging from 35% to 90%. The Ogt methyltransferase provided MFs ranging from 10% to 25%. As was demonstrated by Ada, the Ogt methyltransferase repaired m(6)G poorly in an AXN context. When both methyltransferases were removed, the MF was nearly 100% for all sequence contexts, consistent with the view that the replicative DNA polymerase places T opposite m(6)G during replication irrespective of the local sequence environment.     

Replication inhibition and translesion synthesis on templates containing site-specifically placed cis-diamminedichloroplatinum(II) DNA adducts.

A series of site-specifically plantinated, covalently closed circular M13 genomes (7250 bp) was constructed in order to evaluate the consequences of DNA template damage induced by the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP). Here are reported the synthesis and characterization of genomes containing the intrastrand cross-linked adducts cis-[Pt(NH3)2[d(ApG)-N7(1),-N7(2)]], cis-[Pt-(NH3)2[d(GpCpG)-N7(1),-N7(3)]], and trans-[Pt(NH3)2[d(CpGpCpG)-N3(1),-N7(4)]]. These constructs, as well as the previously reported M13 genome containing a site-specifically placed cis-[Pt(NH3)2[d-(GpG)-N7(1),-N7(2)]] adduct, were used to study replication in vitro. DNA synthesis was initiated from a position approximately 177 nucleotides 3' to the individual adducts, and was terminated either by the adducts or by the end of the template, located approximately 25 nucleotides on the 5' side of the adducts. Analysis of the products of these reactions by gel electrophoresis revealed that, on average, bypass of the cis-DDP adducts occurred approximately 10% of the time and that the cis-[Pt(NH3)2[d(GpG)-N7(1),-N7(2)]] intrastrand cross-link is the most inhibitory lesion. The cis-[Pt(NH3)2[(GpCpG)-N7(1),-N7(3)]] adduct allowed a higher frequency of such translesion synthesis (ca. 25%) for two of the polymerases studied, modified bacteriophage T7 polymerase and Escherichia coli DNA polymerase I (Klenow fragment). These enzymes have either low (Klenow) or no (T7) associated 3' to 5' exonuclease activity. Bacteriophage T4 DNA polymerase, which has a very active 3' to 5' exonuclease, was the most strongly inhibited by all three types of cis-DDP adducts, permitting only 2% translesion synthesis. This enzyme is therefore recommended for replication mapping studies to detect the location of cis-DDP-DNA adducts in a heterologous population. The major replicative enzyme of E. coli, the DNA polymerase III holoenzyme, allowed less than 10% adduct bypass. Postreplication restriction enzyme cleavage studies established that the templates upon which translesion synthesis was observed contained platinum adducts, ruling out the possibility that the observed products were due to a small amount of contamination with unplatinated DNA. The effects on in vitro replication of a recently characterized adduct of trans-DDP [Comess, K. M., Costello, C. E., & Lippard, S. J. (1990) Biochemistry 29, 2102-2110] were also evaluated. This adduct provided a poor block both to DNA polymerases and to restriction enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)     

The distribution and properties of RNA primed initiation sites of DNA synthesis at the replication origin of Escherichia coli chromosome.

RNA-linked DNA molecules were obtained from E. coli dnaCts cells synchronously initiating a new round of chromosome replication. The deoxynucleotides at the transition from primer RNA to DNA were 32P-labeled, and their positions were located on the nucleotide sequence of 1.4 kb genomic region (position -906 to +493) including the oriC and its leftside flanking region. In the r-strand (the counterclockwise strand), many strong transition sites were mapped in the left half portion of the oriC and a few weak sites in the left outside region. In the 1-strand (the clockwise strand), no transition sites were found inside the oriC but many weak sites were found in the left outside region. The results support the initiation mechanism in which the first leading strand synthesis starts with the r-strand counterclockwise from the oriC that is followed by the 1-strand synthesis on the displaced template strand on the left of oriC. Primer RNA molecules attached to the strong r-strand transition sites were only a few residues in length. Properties of the transition sites were discussed.     

Double-strand cleavage and strand joining by the replication initiator protein of filamentous phage f1

The replication initiator protein (gene II protein (gpII] of bacteriophage f1 is a multifunctional protein that plays central roles in initiation and termination of phage DNA replication. It introduces a nick at a specific site on the (+)-strand of supercoiled replicative form DNA. The 3'-hydroxyl end of the nick serves as the primer for (+)-strand rolling-circle replication. Upon completion of a round of synthesis, gpII cleaves and circulaizes the displaced single strand. When Mn2+ is included in the buffer instead of Mg2+, gpII cleaves both strands. In this paper, we investigate the mechanism of the Mn2+-dependent double-strand cleavage activity of gpII. This reaction, unlike nicking in the presence of Mg2+, does not require superhelicity. The reaction proceeds in two kinetic steps: first nicking of the (+)-strand, and then cleavage of the (-)-strand. The nucleotide sequence requirement for nicking is reduced compared to that in the presence of Mg2+. The product of the double-strand cleavage has an unusual structure. The left end is a telomere-like hairpin since the (+)- and (-)-strands are joined, as demonstrated by base sequencing. The right end has a onebase 3'-overhang. This reaction probably reflects the cleavage-joining activity of gpII in the termination event.     

Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus.

Hepatitis C virus (HCV) is the major etiological agent of non-A, non-B post-transfusion hepatitis. Its genome, a (+)-stranded RNA molecule of approximately 9.4 kb, encodes a large polyprotein that is processed by viral and cellular proteases into at least nine different viral polypeptides. As with other (+)-strand RNA viruses, the replication of HCV is thought to proceed via the initial synthesis of a complementary (-) RNA strand, which serves, in turn, as a template for the production of progeny (+)-strand RNA molecules. An RNA-dependent RNA polymerase has been postulated to be involved in both of these steps. Using the heterologous expression of viral proteins in insect cells, we present experimental evidence that an RNA-dependent RNA polymerase is encoded by HCV and that this enzymatic activity is the function of the 65 kDa non-structural protein 5B (NS5B). The characterization of the HCV RNA-dependent RNA polymerase product revealed that dimer-sized hairpin-like RNA molecules are generated in vitro, indicating that NS5B-mediated RNA polymerization proceeds by priming on the template via a 'copy-back' mechanism. In addition, the purified HCV NS5B protein was shown to perform RNA- or DNA oligonucleotide primer-dependent RNA synthesis on templates with a blocked 3' end or on homopolymeric templates. These results represent a first important step towards a better understanding of the life cycle of the HCV.     

Mutagenesis by acrolein-derived propanodeoxyguanosine adducts in human cells

Acrolein, which is widely spread in the environment and is produced by lipid peroxidation in cells, reacts with DNA to form two exocyclic 1,N2-propanodeoxyguanosine (PdG) adducts. To establish their relative contribution to the acrolein mutagenicity, the genotoxic properties of alpha-OH-PdG and gamma-OH-PdG together with their model DNA adduct, PdG, were studied in human cells. DNA adducts were incorporated site-specifically into a SV40/BK virus origin-based shuttle vector and replicated in xeroderma pigmentosum complementation group A (XPA) cells. Analysis of progeny plasmid revealed that alpha-OH-PdG and PdG strongly block DNA synthesis and that both adducts induced base substitutions with G --> T transversions predominating. Primer extension studies, catalyzed by the 3'-->5' exonuclease-deficient Klenow fragment of Escherichia coli pol I, revealed limited extension from the 3' primer termini opposite these two adducts. In contrast, gamma-OH-PdG did not strongly block DNA synthesis or miscode in XPA cells. Primer extension from a dC terminus opposite gamma-OH-PdG was much more efficient than that opposite alpha-OH-PdG or PdG. These results indicate that the minor alpha-OH-PdG adduct is more genotoxic than the major gamma-OH-PdG. Furthermore, experiments using a HeLa whole cell extract indicate that all three DNA adducts are not efficiently removed from DNA by base excision repair.     

Differential DNA recognition and cleavage by EcoRI dependent on the dynamic equilibrium between the two forms of the malondialdehyde-deoxyguanosine adduct

DNA damage may alter the outcome of protein-nucleic acid interactions. The malondialdehyde-deoxyguanosine adduct, 3-(2'-deoxy-beta-d-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin-10-(3H)-one (M(1)dG), miscodes in vivo and in vitro. M(1)dG is an exocyclic adduct that undergoes ring-opening in duplex DNA to form the acyclic adduct, N(2)-(3-oxo-1-propenyl)-deoxyguanosine (N(2)-OPdG). These two adducts have different effects on DNA polymerase bypass and may affect other DNA processing enzymes. We employed the EcoRI restriction endonuclease as a model for the interaction of DNA binding proteins with adducted DNA substrates. The presence of M(1)dG in the EcoRI recognition sequence impaired the ability of the enzyme to cleave DNA, resulting in only 60% cleavage of the adducted strand and 75% cleavage of the complementary strand. Three adducts of similar structure to M(1)dG that are unable to ring-open were cleaved poorly, or not at all, by EcoRI. None of the adducts appeared to inactivate or sequester EcoRI. Additional studies with BssHII and PauI confirmed these results and demonstrated a positional effect of M(1)dG on cleavage efficiency. These data suggest dissimilar modes of protein-nucleic acid interactions based on differences in adduct structure. Comparison of the solution structures of DNA adducts and the crystal structure of EcoRI complexed to substrate suggest a model to explain the functional differences.     

Repair of thymine.guanine and uracil.guanine mismatched base-pairs in bacteriophage M13mp18 DNA heteroduplexes

Repair of thymine.guanine (T.G) and uracil.guanine (U.G) mismatched base-pairs in bacteriophage M13mp18 replicative form (RF) DNA was compared upon transfection into repair-proficient or repair-deficient Escherichia coli strains. Oligonucleotide-directed mutagenesis was used to prepare covalently closed circular heteroduplexes that contained the mismatched base-pair at a restriction recognition site. The heteroduplexes were unmethylated at dam (5'-GATC-3') sites to avoid methylation-directed biasing of repair. In an E. coli host containing uracil-DNA glycosylase (ung+), about 97% of the transfecting U.G-containing heteroduplexes had the U residue excised by the uracil-excision repair system. With the analogous T.G mispair, mismatch repair operated on almost all of the transfecting heteroduplexes and removed the T residue in about 75% of them when the mismatched T was on the minus strand of the RF DNA. Similar preferential excision of the minus-strand's mismatched base was observed whether the heteroduplex RF DNA molecules had only one or both strands unmethylated at dcm (5'-CC(A/T)GG-3') sites and whether the RF DNA was prepared by primer extension in vitro or by reannealing mutant and non-mutant DNA strands. Also, the extent and directionality of repair was the same at a U.G mispair in ung- host cells as at the analogous T.G mispair in ung- or ung+ cells. Only in a mismatch repair-deficient (mutH-) host was the plus strand of the transfecting M13mp18 heteroduplex DNA preferentially repaired. It is suggested that the plus strand nick made by the M13-encoded gene II protein might be employed by a mutH- host to initiate repair on that strand.     

Comparative study of mutagenesis by O6-methylguanine in the human Ha-ras oncogene in E. coli and in vitro.

Single residues of O6-methylguanine (O6-meG) were introduced into the first or second position of codon 12 (GGC; positions 12G1 or 12G2, respectively) or the first position of codon 13 (GGT; position 13G1) of the human Ha-ras oncogene in phage M13-based vectors. After transformation of E.coli, higher mutant plaque frequencies (MPF) were observed at 12G1 and 13G1 than at 12G2 if O6-alkylguanine-DNA alkyltransferase (AGT) had been depleted, while similar MPF were observed at all three positions in the presence of active AGT. Taken together, these observations suggest reduced AGT repair at 12G2. Kinetic analysis of in vitro DNA replication in the same sequences using E. coli DNA polymerase I (Klenow fragment) indicated that variation in polymerase fidelity may contribute to the overall sequence specificity of mutagenesis. By constructing vectors which direct methyl-directed mismatch repair to the (+) or the (-) strand and comparing the MPF values in bacteria proficient or deficient in mismatch repair and/or AGT, it was concluded that, while mutS-mediated mismatch repair did not remove O6-meG from O6-meG:C pairs, this repair mechanism can affect O6-meG mutagenesis by repairing G:T pairs generated through AGT-induced demethylation of O6-meG:T replication intermediates.     

Structural basis of accurate replication beyond a bulky major benzo[a]pyrene adduct by human DNA polymerase kappa

Human Y-family DNA polymerase kappa (polκ) is specialized to bypass bulky lesions in DNA in an error-free way, thus protecting cells from carcinogenic bulky DNA adducts. Benzo[a]pyrene (BP) is one of the most ubiquitous polycyclic aromatic hydrocarbons and an environmental carcinogen. BP covalently modifies DNA and generates mutagenic, bulky adducts. The major BP adduct formed in cells is 10S (+)-trans-anti-BP-N²-dG adduct (BP-dG), which is associated with cancer. The molecular mechanism of how polκ replicates BP-dG accurately is not clear. Here we report the structure of polκ captured at the lesion-extension stage: the enzyme is extending the primer strand after the base pair containing the BP-dG adduct in the template strand at the −1 position. Polκ accommodates the BP adduct in the nascent DNA’s minor groove and keeps the adducted DNA helix in a B-form. Two water molecules cover the edge of the minor groove of the replicating base pair (0 position), which is secured by the BP ring in the −1 position in a 5′ orientation. The 5′ oriented BP adduct keeps correct Watson-Crick base pairing in the active site and promotes high fidelity replication. Our structural and biochemical data reveal a unique molecular basis for accurate DNA replication right after the bulky lesion BP-dG.