Effects of benzo[a]pyrene adduct stereochemistry on downstream DNA replication in vitro: evidence for different adduct conformations within the active site of DNA polymerase I (Klenow fragment)

The presence of bulky adducts in DNA is known to interfere with DNA replication not only at the site of the lesion but also at positions up to 5 nucleotides past the adduct location. Kinetic studies of primer extension by exonuclease-deficient E. coli DNA polymerase I (Klenow fragment) (KF) when (+)-trans- or (+)-cis-B[a]P-N(2)-dG adducts were positioned in the double-stranded region of the primer-templates showed that both stereoisomers significantly block downstream replication. However the (+)-cis adduct, which causes a stronger inhibition of the nucleotides insertion across from and immediately past the lesion, affected the downstream replication to a much smaller extent than did the (+)-trans adduct, especially when the B[a]P-modified dG was properly paired with a dC. The effects of mismatches across from the adduct and the sequence context surrounding the adduct were also dependent on the stereochemistry of the B[a]P adduct. Thus, the identity of the nucleotide across from the adduct that provided the best downstream replication was different for the (+)-cis and (+)-trans adducts, a factor that might differentially contribute to the mutagenic bypass of these lesions. These findings provide strong direct evidence that the conformations of the (+)-cis and (+)-trans adducts within the active site of KF are significantly different and probably differentially affect the interactions of the polymerase with the minor groove, thereby leading to different replication trends. The stereochemistry of the adduct was also found to differentially affect the sequence-mediated primer-template misalignments, resulting in different consequences during the bypass of the lesion.     

The stereochemistry of benzo[a]pyrene-2'-deoxyguanosine adducts affects DNA methylation by SssI and HhaI DNA methyltransferases

The biologically most significant genotoxic metabolite of the environmental pollutant benzo[a]pyrene (B[a]P), (+)-7R,8S-diol 9S,10R-epoxide, reacts chemically with guanine in DNA, resulting in the predominant formation of (+)-trans-B[a]P-N(2)-dG and, to a lesser extent, (+)-cis-B[a]P-N(2)-dG adducts. Here, we compare the effects of the adduct stereochemistry and conformation on the methylation of cytosine catalyzed by two purified prokaryotic DNA methyltransferases (MTases), SssI and HhaI, with the lesions positioned within or adjacent to their CG and GCGC recognition sites, respectively. The fluorescence properties of the pyrenyl residues of the (+)-cis-B[a]P-N(2)-dG and (+)-trans-B[a]P-N(2)-dG adducts in complexes with MTases are enhanced, but to different extents, indicating that aromatic B[a]P residues are positioned in different microenvironments in the DNA-protein complexes. We have previously shown that the (+)-trans-isomeric adduct inhibits both the binding and methylating efficiencies (k(cat)) of both MTases [Subach OM, Baskunov VB, Darii MV, Maltseva DV, Alexandrov DA, Kirsanova OV, Kolbanovskiy A, Kolbanovskiy M, Johnson F, Bonala R, et al. (2006) Biochemistry45, 6142-6159]. Here we show that the stereoisomeric (+)-cis-B[a]P-N(2)-dG lesion has only a minimal effect on the binding of these MTases and on k(cat). The minor-groove (+)-trans adduct interferes with the formation of the normal DNA minor-groove contacts with the catalytic loop of the MTases. However, the intercalated base-displaced (+)-cis adduct does not interfere with the minor-groove DNA-catalytic loop contacts, allowing near-normal binding of the MTases and undiminished k(cat) values.     

Effects of benzo[a]pyrene DNA adducts on Escherichia coli DNA polymerase I (Klenow fragment) primer-template interactions: evidence for inhibition of the catalytically active ternary complex formation

Benzo[a]pyrene diol epoxide (B[a]PDE) adducts are strong blocks of DNA replication in vitro, allowing the rare incorporation of a nucleotide across from the lesion and negligibly small extent of further bypass. To study the mechanistic details of this process, a gel-retardation assay was used to measure the dissociation constants for the binding of DNA polymerase I (Klenow fragment) (KF) to the primer-templates containing a (+)-trans- or (+)-cis-B[a]P-N(2)-dG adduct. When the primer was terminated one nucleotide before the adduct, the presence of a (+)-trans-B[a]P-N(2)-dG adduct did not affect the binding while a (+)-cis-B[a]P-N(2)-dG adduct caused a slight decrease in affinity. The presence of any dNTP decreased the affinity of KF to the modified primer-templates. (In contrast, a strong increase of the affinity to unmodified primer-templates was observed in the presence of the next correct dNTP.) Limited protease digestion experiments indicated that a closed ternary complex of KF with the modified primer-templates was not detectable in the presence of any dNTP, whereas it was clearly observed with unmodified template in the presence of the next correct nucleotide. These findings suggest that these adducts may interfere with the conformational change to the catalytically active closed ternary complex and/or cause significant destabilization of this complex. When the primers extended to the position across from the adduct, the affinity of KF was significantly decreased irrespective of the identity of the base across from the adduct, possibly explaining the low bypass of the lesion. Interestingly, the stability of these DNA-polymerase complexes correlated with nucleotide insertion kinetics for the unmodified and (+)-trans-B[a]PDE-modified templates.     

Molecular modeling of the major benzo[a]pyrene N2-dG adduct in cases where mutagenesis results are known in double stranded DNA

The potent mutagen/carcinogen benzo[a]pyrene (B[a]P) is metabolically activated to (+)-anti-B[a]PDE, which induces a full spectrum of mutations (e.g. GC-->TA, GC-->AT, etc.). One hypothesis for this complexity is that different mutations are induced by different conformations of its major adduct [+ta]-B[a]P-N2-dG when bypassed during DNA replication (probably by different DNA polymerases). Previous molecular modeling studies suggested that B[a]P-N2-dG adducts can in principle adopt at least 16 potential conformational classes in ds-DNA. Herein we report on molecular modeling studies with the eight conformations most likely to be relevant to base substitution mutagenesis in 10 cases where mutagenesis has been studied in ds-DNA plasmids in E. coli with B[a]P-N2-dG adducts of differing stereoisomers and DNA sequence contexts, as well as in five cases where the conformation is known by NMR. Of the approximately 11,000 structures generated in this study, the computed lowest energy structures are reported for 120 cases (i.e. eight conformations and 15 examples), and their conformations compared. Of the eight conformations, four are virtually always computed to be high in energy. The remaining four lower energy conformations include two with the BP moiety in the minor groove (designated: BPmi5 and BPmi3), and two base-displaced conformations, one with the dG moiety in the major groove (designated: Gma5) and one with the dG in the minor groove (designated: Gmi3). Interestingly, these four are the only conformations that have been observed for B[a]P-N2-dG adducts in NMR studies. Independent of sequence contexts and adduct stereochemistry, BPmi5 structures tend to look reasonably similar, as do BPmi3 structures, while the base-displaced structures Gma5 and BPmi3 tend to show greater variability in structure. A correlation was sought between modeling and mutagenesis results in the case of the low energy conformations BPmi5, BPmi3, Gma5 and Gma3. Plots of log[(G-->T)/(G-->A)] versus energy[(conformation X)-(conformation Y)] were constructed for all six pairwise combinations of these four conformations, and the only plot giving a straight line involved Gma5 and Gmi3. While this finding is striking, its significance is unclear (as discussed).     

Toward an understanding of the role of DNA adduct conformation in defining mutagenic mechanism based on studies of the major adduct (formed at N(2)-dG) of the potent environmental carcinogen, benzo[a]pyrene

The process of carcinogenesis is initiated by mutagenesis, which often involves replication past damaged DNA. One question - what exactly is a DNA polymerase seeing when it incorrectly copies a damaged DNA base (e.g., inserting dATP opposite a dG adduct)? - has not been answered in any case. Herein, we reflect on this question, principally by considering the mutagenicity of one activated form of benzo[a]pyrene, (+)-anti-B[a]PDE, and its major adduct [+ta]-B[a]P-N(2)-dG. In previous work, [+ta]-B[a]P-N(2)-dG was shown to be capable of inducing>95% G-->T mutations in one sequence context (5'-TGC), and approximately 95% G-->A mutations in another (5'-AGA). This raises the question - how can a single chemical entity induce different mutations depending upon DNA sequence context? Our current working hypothesis is that adduct conformational complexity causes adduct mutational complexity, where DNA sequence context can affect the former, thereby influencing the latter. Evidence supporting this hypothesis was discussed recently (Seo et al., Mutation Res. [in press]). Assuming this hypothesis is correct (at least in some cases), one goal is to consider what these mutagenic conformations might be. Based on molecular modeling studies, 16 possible conformations for [+ta]-B[a]P-N(2)-dG are proposed. A correlation between molecular modeling and mutagenesis work suggests a hypothesis (Hypothesis 3): a base displaced conformation with the dG moiety of the adduct in the major vs. minor groove gives G-->T vs. G-->A mutations, respectively. (Hypothesis 4, which is a generalized version of Hypothesis 3, is also proposed, and can potentially rationalize aspects of both [+ta]-B[a]P-N(2)-dG and AP-site mutagenesis, as well as the so-called "A-rule".) Finally, there is a discussion of how conformational complexity might explain some unusual mutagenesis results that suggest [+ta]-B[a]P-N(2)-dG can become trapped in different conformations, and why we think it makes sense to interpret adduct mutagenesis results by modeling ds-DNA (at least in some cases), even though the mutagenic event must occur at a ss/ds-DNA junction in the presence of a DNA polymerase.     

Impact of benzo[a]pyrene-2'-deoxyguanosine lesions on methylation of DNA by SssI and HhaI DNA methyltransferases

DNA damage caused by the binding of the tumorigen 7R,8S-diol 9S,10R-epoxide (B[a]PDE), a metabolite of bezo[a]pyrene, to guanine in CpG dinucleotide sequences could affect DNA methylation and, thus, represent a potential epigenetic mechanism of chemical carcinogenesis. In this work, we investigated the impact of stereoisomeric (+)- and (-)-trans-anti-B[a]P-N(2)-dG adducts (B(+) and B(-)) on DNA methylation by prokaryotic DNA methyltransferases M.SssI and M.HhaI. These two methyltransferases recognize CpG and GCGC sequences, respectively, and transfer a methyl group to the C5 atom of cytosine (C). A series of 18-mer unmethylated or hemimethylated oligodeoxynucleotide duplexes containing trans-anti-B[a]P-N(2)-dG adducts was generated. The B(+) or B(-) residues were introduced either 5' or 3' adjacent or opposite to the target 2'-deoxycytidines. The B[a]PDE lesions practically produced no effect on M.SssI binding to DNA but reduced M.HhaI binding by 1-2 orders of magnitude. In most cases, the benzo[a]pyrenyl residues decreased the methylation efficiency of hemimethylated and unmethylated DNA by M.SssI and M.HhaI. An absence of the methylation of hemimethylated duplexes was observed when either the (+)- or the (-)-trans-anti-B[a]P-N(2)-dG adduct was positioned 5' to the target dC. The effects observed may be related to the minor groove conformation of the bulky benzo[a]pyrenyl residue and to a perturbation of the normal contacts of the methyltransferase catalytic loop with the B[a]PDE-modified DNA. Our results indicate that a trans-anti-B[a]P-N(2)-dG lesion flanking a target dC in the CpG dinucleotide sequence on its 5'-side has a greater adverse impact on methylation than the same lesion when it is 3' adjacent or opposite to the target dC.     

Mirror image stereoisomers of the major benzo[a]pyrene N2-dG adduct are bypassed by different lesion-bypass DNA polymerases in E. coli

The potent mutagen/carcinogen benzo[a]pyrene (B[a]P) is metabolically activated to (+)-anti-B[a]PDE, which induces a full spectrum of mutations (e.g., G-to-T, G-to-A, -1 frameshifts, etc.) via its major adduct [+ta]-B[a]P-N2-dG. We recently showed that the dominant G-to-T mutation depends on DNA polymerase V (DNAP V), but not DNAPs IV or II, when studied in a 5'-TG sequence in E. coli. Herein we investigate what DNAPs are responsible for non-mutagenic bypass with [+ta]-B[a]P-N2-dG, along with its mirror image adduct [-ta]-B[a]P-N2-dG. Each adduct is built into a 5'-TG sequence in a single stranded M13 phage vector, which is then transformed into eight different E. coli strains containing all combinations of proficiency and deficiency in the three lesion-bypass DNAPs II, IV and V. Based on M13 progeny output, non-mutagenic bypass with [-ta]-B[a]P-N2-dG depends on DNAP IV. In contrast, non-mutagenic bypass with [+ta]-B[a]P-N2-dG depends on both DNAPs IV and V, where arguments suggest that DNAP IV is involved in dCTP insertion, while DNAP V is involved in extension of the adduct-G:C base pair. Numerous findings indicate that DNAP II has a slight inhibitory effect on the bypass of [+ta]- and [-ta]-B[a]P-N2-dG in the case of both DNAPs IV and V. In conclusion, for efficient non-mutagenic bypass (dCTP insertion) in E. coli, [+ta]-B[a]P-N2-dG requires DNAPs IV and V, [-ta]-B[a]P-N2-dG requires only DNAP IV, while DNAP II is inhibitory to both, and experiments to investigate these differences should provide insights into the mechanism and purpose of these lesion-bypass DNAPs.     

A role for DNA polymerase V in G --> T mutations from the major benzo[a]pyrene N2-dG adduct when studied in a 5'-TGT sequence in E. coli

Benzo[a]pyrene (B[a]P), a potent mutagen/carcinogen, is metabolically activated to (+)-anti-B[a]PDE, which induces a full spectrum of mutations (e.g. GC --> TA, GC --> AT, etc.) principally via its major adduct [+ta]-B[a]P-N2-dG. Recent findings suggest that different lesion bypass DNA polymerases may be involved in different mutagenic pathways, which is the subject of this report. [+ta]-B[a]P-N2-dG built into a plasmid in a 5'-TGT sequence gives approximately equal numbers of G --> T and G --> A mutations when host E. coli are UV irradiated prior to transformation, so this sequence context was chosen to investigate what DNA polymerases are involved in G --> T versus G --> A mutations. G --> T mutations decline (>10-fold) if E. coli either are not UV-irradiated or are deficient in DNA polymerase V ((delta)umuD/C), demonstrating a role for damage-inducible DNA Pol V in a G --> T pathway. G --> T mutations are not affected by transformation into E. coli deficient in either DNA polymerases II or IV. While the work herein was in progress, Lenne-Samuel et al. [Mol. Microbiol. 38 (2000) 299] built the same adduct into a plasmid in a 5'-GGA sequence, and showed that the frequency of G --> T mutations was similar in UV-irradiated and unirradiated host E. coli cells, suggesting no involvement by damage-inducible, lesion bypass DNA polymerases (i.e., not II, IV or V); furthermore, a role for DNA Pol V was explicitly ruled out. The easiest way to reconcile the findings of Lenne-Samuel et al. with the findings herein is if two G --> T mutagenic pathways exist for [+ta]-B[a]P-N2-dG, where sequence context dictates which pathway is followed. In contrast to the G --> T mutations, herein G --> A mutations from [+ta]-B[a]P-N2-dG in the 5'-TGT sequence context are shown not to be affected by UV-irradiation of host E. coli, and are not dependent on DNA Pol V, or Pol II, Pol IV, or the damage-inducible, but SOS-independent UVM system. Published studies, however, have shown that G --> A mutations are usually enhanced by UV-irradiation of host E. coli prior to the introduction of plasmids either site-specifically modified with [+ta]-B[a]P-N2-dG or randomly adducted with (+)-anti-B[a]PDE; both findings imply the involvement of a lesion-bypass DNA polymerase. These disparate results suggest the existence of two G --> A mutagenic pathways for [+ta]-B[a]P-N2-dG as well, although confirmation of this awaits further study. In conclusion, a comparison between the evidence presented herein and published findings suggests the existence of two distinct mutagenic pathways for both G --> T and G --> A mutations from [+ta]-B[a]P-N2-dG, where in each case one pathway is not damage-inducible and not dependent on a lesion-bypass DNA polymerase, while the second pathway is damage-inducible and dependent on a lesion-bypass DNA polymerase. Furthermore, DNA sequence context appears to dictate which pathway (as defined by the involvement of different DNA polymerases) is followed in each case.     

Extending the understanding of mutagenicity: structural insights into primer-extension past a benzo[a]pyrene diol epoxide-DNA adduct

DNA polymerase enzymes employ a number of innate fidelity mechanisms to ensure the faithful replication of the genome. However, when confronted with DNA damage, their fidelity mechanisms can be evaded, resulting in a mutation that may contribute to the carcinogenic process. The environmental carcinogen benzo[a]pyrene is metabolically activated to reactive intermediates, including the tumorigenic (+)-anti-benzo[a]pyrene diol epoxide, which can attack DNA at the exocyclic amino group of guanine to form the major (+)-trans-anti-[BP]-N(2)-dG adduct. Bulky adducts such as (+)-trans-anti-[BP]-N(2)-dG primarily block DNA replication, but are occasionally bypassed and cause mutations if paired with an incorrect base. In vitro standing-start primer-extension assays show that the preferential insertion of A opposite (+)-trans-anti-[BP]-N(2)-dG is independent of the sequence context, but the primer is extended preferentially when dT is positioned opposite the damaged base in a 5'-CG*T-3' sequence context. Regardless of the base positioned opposite (+)-trans-anti-[BP]-N(2)-dG, extension of the primer past the lesion site poses the greatest block to polymerase progression. In order to gain insight into primer-extension of each base opposite (+)-trans-anti-[BP]-N(2)-dG, we carried out molecular modeling and 1.25 ns unrestrained molecular dynamics simulations of the adduct in the +1 position of the template within the replicative pol I family T7 DNA polymerase. Each of the four bases was modeled at the 3' terminus of the primer, incorporated opposite the adduct, and the next-to-be replicated base was in the active site with its Watson-Crick partner as the incoming nucleotide. As in our studies of nucleotide incorporation, (+)-trans-anti-[BP]-N(2)-dG was modeled in the syn conformation in the +1 position, with the BP moiety on the open major groove side of the primer-template duplex region, leaving critical protein-DNA interactions intact. The present work revealed that the efficiency of primer-extension past this bulky adduct opposite each of the four bases in the 5'-CG*T-3' sequence can be rationalized by the stability of interactions between the polymerase protein, primer-template DNA and incoming nucleotide. However, the relative stabilization of each nucleotide opposite (+)-trans-anti-[BP]-N(2)-dG in the +1 position (T > G > A > or = C) differed from that when the adduct and partner were the nascent base-pair (A > T > or = G > C). In addition, extension past (+)-trans-anti-[BP]-N(2)-dG may pose a greater block to a high fidelity DNA polymerase than does nucleotide incorporation opposite the adduct because the presence of the modified base-pair in the +1 position is more disruptive to the polymerase-DNA interactions than it is within the active site itself. The dN:(+)-trans-anti-[BP]-N(2)-dG base-pair is strained to shield the bulky aromatic BP moiety from contact with the solvent in the +1 position, causing disruption of protein-DNA interactions that would likely result in decreased extension of the base-pair. These studies reveal in molecular detail the kinds of specific structural interactions that determine the function of a processive DNA polymerase when challenged by a bulky DNA adduct.     

Factors that influence the mutagenic patterns of DNA adducts from chemical carcinogens

Carcinogens are generally mutagens, which is understandable given that tumor cells grow uncontrollably because they have mutations in critical genes involved in growth control. Carcinogens often induce a complex pattern of mutations (e.g., GC-->TA, GC-->AT, etc.). These mutations are thought to be initiated when a DNA polymerase encounters a carcinogen-DNA adduct during replication. In principle, mutational complexity could be due to either a collection of different adducts each inducing a single kind of mutation (Hypothesis 1a), or a single adduct inducing different kinds of mutations (Hypothesis 1b). Examples of each are discussed. Regarding Hypothesis 1b, structural factors (e.g., DNA sequence context) and biological factors (e.g., differing DNA polymerases) that can affect the pattern of adduct mutagenesis are discussed. This raises the question: how do structural and biological factors influence the pattern of adduct mutagenesis. For structural factors, three possibilities are considered: (Hypothesis 2a) a single conformation of an adduct giving rise to multiple mutations -- dNTP insertion by DNA polymerase being influenced by (e.g.) the surrounding DNA sequence context; (Hypothesis 2b) a variation on this ("dislocation mutagenesis"); or (Hypothesis 2c) a single adduct adopting multiple conformations, each capable of giving a different pattern of mutations. Hypotheses 2a, 2b and 2c can each in principle rationalize many mutational results, including how the pattern of adduct mutagenesis might be influenced by factors, such as DNA sequence context. Five lines of evidence are discussed suggesting that Hypothesis 2c can be correct for base substitution mutagenesis. For example, previous work from our laboratory was interpreted to indicate that [+ta]-B[a]P-N(2)-dG in a 5'-CGG sequence context (G115) could be trapped in a conformation giving predominantly G-->T mutations, but heating caused the adduct to equilibrate to its thermodynamic mixture of conformations, leading to a decrease in the fraction of G-->T mutations. New work is described suggesting that [+ta]-B[a]P-N(2)-dG at G115 can also be trapped predominantly in the G-->A mutational conformation, from which equilibration can also occur, leading to an increase in the fraction of G-->T mutations. Evidence is also presented that the fraction of G-->T mutations is higher when [+ta]-B[a]P-N(2)-dG at G115 is in ss-DNA ( approximately 89%) vs. ds-DNA ( approximately 66%), a finding that can be rationalized if the mixture of adduct conformations is different in ss- and ds-DNA. In summary, the factors affecting adduct mutagenesis are reviewed and five lines of evidence that support one hypothesis (2c: adduct conformational complexity can cause adduct mutational complexity) are discussed.     

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.     

Differential incision of bulky carcinogen-DNA adducts by the UvrABC nuclease: comparison of incision rates and the interactions of Uvr subunits with lesions of different structures

The UvrABC nuclease system from Escherichia coli removes DNA damages induced by a wide range of chemical carcinogens with variable efficiencies. The interactions with UvrABC proteins of the following three lesions site-specifically positioned in DNA, and of known conformations, were investigated: (i) adducts derived from the binding of the (-)-(7S,8R,9R,10S) enantiomer of 7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-anti-BPDE] by cis-covalent addition to N(2)-2'-deoxyguanosine [(-)-cis-anti-BP-N(2)-dG], (ii) an adduct derived from the binding of the (+)-(1R,2S,3S,4R) enantiomer of 1,2-dihydroxy-3,4-epoxy-1,2,3, 4-tetrahydro-5-methylchrysene [(+)-anti-5-MeCDE] by trans addition to N(2)-2'-deoxyguanosine [(+)-trans-anti-MC-N(2)-dG], and (iii) a C8-2'-deoxyguanosine adduct (C8-AP-dG) formed by reductively activated 1-nitropyrene (1-NP). The influence of these three different adducts on UvrA binding affinities, formation of UvrB-DNA complexes by quantitative gel mobility shift analyses, and the rates of UvrABC incision were investigated. The binding affinities of UvrA varied among the three adducts. UvrA bound to the DNA adduct (+)-trans-anti-MC-N(2)-dG with the highest affinity (K(d) = 17 +/- 2 nM) and to the DNA containing C8-AP-dG with the least affinity (K(d) = 28 +/- 1 nM). The extent of complex formation with UvrB was also the lowest with the C8-AP-dG adduct. 5' Incisions occurred at the eighth phosphate from the modified guanine. The major 3' incision site corresponded to the fifth phosphodiester bond for all three adducts. However, additional 3' incisions were observed at the fourth and sixth phosphates in the case of the C8-AP-dG adduct, whereas in the case of the (-)-cis-anti-BP-N(2)-dG and (+)-trans-anti-MC-N(2)-dG lesions additional 3' cleavage occurred at the sixth and seventh phosphodiester bonds. Both the initial rate and the extent of 5' and 3' incisions revealed that C8-AP-dG was repaired less efficiently in comparison to the (-)-cis-anti-BP-N(2)-dG and (+)-trans-anti-MC-N(2)-dG containing DNA adducts. Our study showed that UvrA recognizes conformational changes induced by structurally different lesions and that in certain cases the binding affinities of UvrA and UvrB can be correlated with the incision rates. The size of the bubble formed around the damaged site with mismatched bases also appears to influence the incision rates. A particularly noteworthy finding in this study is that UvrABC repair of a substrate with no base opposite C8-AP-dG was quite inefficient as compared to the same adduct with a C opposite it. These findings are discussed in terms of the available NMR solution structures.     

Toward understanding the mutagenicity of an environmental carcinogen: structural insights into nucleotide incorporation preferences

Bulky carcinogen-DNA adducts, including (+)-trans-anti-[BP]-N(2)-dG derived from the reaction of (+)-anti-benzo[a]pyrene diol epoxide with guanine, often block the progression of DNA polymerases. However, when rare bypass of the lesions does occur, they may be misreplicated. Experimental results have shown that nucleotides are inserted opposite the (+)-trans-anti-[BP]-N(2)-dG adduct by bacteriophage T7 DNA polymerase with the order of preference A>T>or=G>C. To gain structural insights into the effects of the bulky adduct on nucleotide incorporation within the polymerase active site, molecular modeling and molecular dynamics simulations were carried out using T7 DNA polymerase to permit the relation of function to structure. We modeled the (+)-trans-anti-[BP]-N(2)-dG adduct opposite incoming dGTP, dTTP and dCTP nucleotides, as well as unmodified guanine opposite its normal partner dCTP as a control, to compare with our previous simulation with dATP opposite the adduct. The modeling required that the (+)-trans-anti-[BP]-N(2)-dG adduct adopt the syn conformation in each case to avoid deranging essential protein-DNA interactions. While the dATP: (+)-trans-anti-[BP]-N(2)-dG pair was well accommodated within the active site of T7 DNA polymerase, dCTP fit poorly opposite the adduct, adopting an orientation perpendicular to the plane of the syn modified guanine during the simulation. Rotation about the glycosidic bond of the dCTP residue to this abnormal position was allowed because only one hydrogen bond between dCTP and the (+)-trans-anti-[BP]-N(2)-dG residue evolved during the simulation, and this hydrogen bond was directly across from the dCTP glycosidic bond. The dTTP and dGTP nucleotides, incorporated with an intermediate preference opposite (+)-trans-anti-[BP]-N(2)-dG, were accommodated reasonably well, but not as stably as the dATP nucleotide, due to a skewed primer-template alignment and more exposed BP moiety, respectively. In addition, the extent of stabilizing interactions between the nascent base-pair in each simulation was correlated positively with the incorporation preference of that particular nucleotide. The dATP nucleotide is accommodated most stably opposite the adduct, with protein-DNA hydrogen bonding interactions and an active-site pocket size that do not deviate significantly from those of the control simulation. The simulations of dTTP and dGTP opposite (+)-trans-anti-[BP]-N(2)-dG exhibited more instability in interactions between the protein and the nascent base-pair than the dATP system. However, the active-site pocket size of the dTTP and dGTP simulations remained stable. The dCTP: (+)-trans-anti-[BP]-N(2)-dG system had the least number of stabilizing interactions, and the active-site pocket of this system increased in size significantly compared to the control and other dNTPs opposite the adduct. These simulations elucidated why A is inserted opposite (+)-trans-anti-[BP]-N(2)-dG most frequently, while T and G are inserted opposite the adduct to an extent intermediate between A and C, and C is most rarely incorporated. Structural rationalization of the incorporation preference opposite (+)-trans-anti-[BP]-N(2)-dG by T7 DNA polymerase contributes to providing a molecular explanation for mutations caused by this carcinogen-DNA adduct in a model system.     

Base pair conformation-dependent excision of benzo[a]pyrene diol epoxide-guanine adducts by human nucleotide excision repair enzymes.

Human nucleotide excision repair processes carcinogen-DNA adducts at highly variable rates, even at adjacent sites along individual genes. Here, we identify conformational determinants of fast or slow repair by testing excision of N2-guanine adducts formed by benzo[a]pyrene diol epoxide (BPDE), a potent and ubiquitous mutagen that induces mainly G x C-->T x A transversions and frameshift deletions. We found that human nucleotide excision repair processes the predominant (+)-trans-BPDE-N2-dG adduct 15 times less efficiently than a standard acetylaminofluorene-C8-dG lesion in the same sequence. No difference was observed between (+)-trans- and (-)-trans-BPDE-N2-dG, but excision was enhanced about 10-fold by changing the adduct configurations to either (+)-cis- or (-)-cis-BPDE-N2-dG. Conversely, excision of (+)-cis- and (-)-cis- but not (+)-trans-BPDE-N2-dG was reduced about 10-fold when the complementary cytosine was replaced by adenine, and excision of these BPDE lesions was essentially abolished when the complementary deoxyribonucleotide was missing. Thus, a set of chemically identical BPDE adducts yielded a greater-than-100-fold range of repair rates, demonstrating that nucleotide excision repair activity is entirely dictated by local DNA conformation. In particular, this unique comparison between structurally highly defined substrates shows that fast excision of BPDE-N2-dG lesions is correlated with displacement of both the modified guanine and its partner base in the complementary strand from their normal intrahelical positions. The very slow excision of carcinogen-DNA adducts located opposite deletion sites reveals a cellular strategy that minimizes the fixation of frameshifts after mutagenic translesion synthesis.     

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.     

The spacious active site of a Y-family DNA polymerase facilitates promiscuous nucleotide incorporation opposite a bulky carcinogen-DNA adduct: elucidating the structure-function relationship through experimental and computational approaches

Y-family DNA polymerases lack some of the mechanisms that replicative DNA polymerases employ to ensure fidelity, resulting in higher error rates during replication of undamaged DNA templates and the ability to bypass certain aberrant bases, such as those produced by exposure to carcinogens, including benzo[a]pyrene (BP). A tumorigenic metabolite of BP, (+)-anti-benzo-[a]pyrene diol epoxide, attacks DNA to form the major 10S (+)-trans-anti-[BP]-N(2)-dG adduct, which has been shown to be mutagenic in a number of prokaryotic and eukaryotic systems. The 10S (+)-trans-anti-[BP]-N(2)-dG adduct can cause all three base substitution mutations, and the SOS response in Escherichia coli increases bypass of bulky adducts, suggesting that Y-family DNA polymerases are involved in the bypass of such lesions. Dpo4 belongs to the DinB branch of the Y-family, which also includes E. coli pol IV and eukaryotic pol kappa. We carried out primer extension assays in conjunction with molecular modeling and molecular dynamics studies in order to elucidate the structure-function relationship involved in nucleotide incorporation opposite the bulky 10S (+)-trans-anti-[BP]-N(2)-dG adduct by Dpo4. Dpo4 is able to bypass the 10S (+)-trans-anti-[BP]-N(2)-dG adduct, albeit to a lesser extent than unmodified guanine, and the V(max) values for insertion of all four nucleotides opposite the adduct by Dpo4 are similar. Computational studies suggest that 10S (+)-trans-anti-[BP]-N(2)-dG can be accommodated in the active site of Dpo4 in either the anti or syn conformation due to the limited protein-DNA contacts and the open nature of both the minor and major groove sides of the nascent base pair, which can contribute to the promiscuous nucleotide incorporation opposite this lesion.     

Effects of base sequence context on translesion synthesis past a bulky (+)-trans-anti-B[a]P-N2-dG lesion catalyzed by the Y-family polymerase pol kappa

The effects of bases flanking single bulky lesions derived from the binding of a benzo[a]pyrene 7,8-diol 9,10-epoxide derivative ((+)-7R,8S,9S,10R stereoisomer) to N(2)-guanine (G*) on translesion bypass catalyzed by the Y-family polymerase pol kappa (hDinB1) were examined in vitro. The lesions were positioned near the middle of six different 43-mer 5'-...XG*Y... sequences (X, Y = C, T, or G, with all other bases remaining fixed). The complementary dCTP is preferentially inserted opposite G* in all of the sequences; however, the proportions of other dNTPs inserted varies as a function of X and Y. The dCTP insertion efficiencies, f(ins) = (V(max)/K(m))(ins), are smaller in the XG*Y than in XGY sequences by factors of approximately 50-90 (GG*T and GG*C) or 5000-25000 (TG*G and CG*G). Remarkably, in XG*Y sequences, f(ins) varies by as much as 3 orders of magnitude, being smallest with G flanking the lesions on the 3'-side and highest with G flanking the adducts on the 5'-side. One-step primer extension efficiencies just beyond the lesions (f(ext)) are generally smaller than f(ins) and also depend on base sequence. However, reasonably efficient translesion bypass of the (+)-trans-[BP]-N(2)-dG adducts is observed in all sequences in running-start experiments with full, or nearly full, primer extension being observed under conditions of [dNTP] > K(m). The key features here are the relatively robust values of the kinetic parameters V(max) that are either diminished to a moderate extent or even enhanced in the presence of the (+)-trans-[BP]-N(2)-dG adducts. In contrast to the small effects of the lesions on V(max), the apparent K(m) values are orders of magnitude greater in XG*Y than in the unmodified XGY sequences. Thus the bypass of (+)-trans-[BP]-N(2)-dG adducts under conditions when [dNTP] < K(m) is quite inefficient. These considerations may be of importance in vivo where [dNTP] 相似文献   

Robust incision of Benoz[a]pyrene-7,8-dihyrodiol-9,10-epoxide-DNA adducts by a recombinant thermoresistant interspecies combination UvrABC endonuclease system

Prokaryotic DNA repair nucleases are useful reagents for detecting DNA lesions. UvrABC endonuclease, encoded by the UvrA, UvrB, and UvrC genes can incise DNA containing bulky nucleotide adducts and intrastrand cross-links. UvrA, UvrB, and UvrC were cloned from Bacillus caldotenax (Bca)and UvrC from Thermatoga maritima (Tma), and recombinant proteins were overexpressed in and purified from Escherichia coli. Incision activities of UvrABC composed of all Bca-derived subunits (UvrABC(Bca)) and an interspecies combination UvrABC composed of Bca-derived UvrA and UvrB and Tma-derived UvrC (UvrABC(Tma)) were compared on benoz[a]pyrene-7,8-dihyrodiol-9,10-epoxide (BPDE)-adducted substrates. Both UvrABC(Bca) and UvrABC(Tma) specifically incised both BPDE-adducted plasmid DNAs and site-specifically modified 50-bp oligonucleotides containing a single (+)-trans- or (+)-cis-BPDE adduct. Incision activity was maximal at 55-60 degrees C. However, UvrABC(Tma) was more robust than UvrABC(Bca) with 4-fold greater incision activity on BPDE-adducted oligonucleotides and 1.5-fold greater on [(3)H]BPDE-adducted plasmid DNAs. Remarkably, UvrABC(Bca) incised only at the eighth phosphodiester bond 5' to the BPDE-modified guanosine. In contrast, UvrABC(Tma) performed dual incision, cutting at both the fifth phosphodiester bond 3' and eighth phosphodiester bond 5' from BPDE-modified guanosine. BPDE adduct stereochemistry influenced incision activity, and cis adducts on oligonucleotide substrates were incised more efficiently than trans adducts by both UvrABC(Bca) and UvrABC(Tma). UvrAB-DNA complex formation was similar with (+)-trans- and (+)-cis-BPDE-adducted substrates, suggesting that UvrAB binds both adducts equally and that adduct configuration modifies UvrC recognition of the UvrAB-DNA complex. The dual incision capabilities and higher incision activity of UvrABC(Tma) make it a robust tool for DNA adduct studies.     

Hierarchy of DNA damage recognition in Escherichia coli nucleotide excision repair

DNA damage recognition plays a central role in nucleotide excision repair (NER). Here we present evidence that in Escherichia coli NER, DNA damage is recognized through at least two separate but successive steps, with the first focused on distortions from the normal structure of the DNA double helix (initial recognition) and the second specifically recognizing the type of DNA base modifications (second recognition), after an initial local separation of the DNA strands. DNA substrates containing stereoisomeric (+)- or (-)-trans- or (+)- or (-)-cis-BPDE-N(2)-dG lesions in DNA duplexes of known conformations were incised by UvrABC nuclease with efficiencies varying by up to 3-fold. However, these stereoisomeric adducts, when positioned in an opened, single-stranded DNA region, were all incised with similar efficiencies and with enhanced rates (by factors of 1.4-6). These bubble substrates were also equally and efficiently incised by UvrBC nuclease without UvrA. Furthermore, removal of the Watson-Crick partner cytosine residue (leaving an abasic site) in the complementary strand opposite a (+)-cis-BPDE-N(2)-dG lesion led to a significant reduction in both the binding of UvrA and the incision efficiency of UvrABC by a factor of 5. These data suggest that E. coli NER features a dynamic two-stage recognition mechanism.