Mutagenic nucleotide incorporation and hindered translocation by a food carcinogen C8-dG adduct in Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4): modeling and dynamics studies

Bulky carcinogen-DNA adducts commonly cause replicative polymerases to stall, leading to a switch to bypass polymerases. We have investigated nucleotide incorporation opposite the major adduct of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the DinB family polymerase, Dpo4, using molecular modeling and molecular dynamics (MD) simulations. PhIP, the most prevalent heterocyclic aromatic amine formed by cooking of proteinaceous food, is mutagenic in mammalian cells and is implicated in mammary and colon tumors. Our results show that the dG-C8-PhIP adduct can be accommodated in the spacious major groove Dpo4 open pocket, with Dpo4 capable of incorporating dCTP, dTTP or dATP opposite the adduct reasonably well. However, the PhIP ring system on the minor groove side would seriously disturb the active site, regardless of the presence and identity of dNTP. Furthermore, the simulations indicate that dATP and dTTP are better incorporated in the damaged system than in their respective mismatched but unmodified controls, suggesting that the PhIP adduct enhances incorporation of these mismatches. Finally, bulky C8-dG adducts, situated in the major groove, are likely to impede translocation in this polymerase (Rechkoblit et al. (2006), PLoS Biol., 4, e11). However, N(2)-dG adducts, which can reside on the minor groove side, appear to cause less hindrance when in this position

Stepwise Translocation of Dpo4 Polymerase during Error-Free Bypass of an oxoG Lesion

7,8-dihydro-8-oxoguanine (oxoG), the predominant lesion formed following oxidative damage of DNA by reactive oxygen species, is processed differently by replicative and bypass polymerases. Our kinetic primer extension studies demonstrate that the bypass polymerase Dpo4 preferentially inserts C opposite oxoG, and also preferentially extends from the oxoG•C base pair, thus achieving error-free bypass of this lesion. We have determined the crystal structures of preinsertion binary, insertion ternary, and postinsertion binary complexes of oxoG-modified template-primer DNA and Dpo4. These structures provide insights into the translocation mechanics of the bypass polymerase during a complete cycle of nucleotide incorporation. Specifically, during noncovalent dCTP insertion opposite oxoG (or G), the little-finger domain–DNA phosphate contacts translocate by one nucleotide step, while the thumb domain–DNA phosphate contacts remain fixed. By contrast, during the nucleotidyl transfer reaction that covalently incorporates C opposite oxoG, the thumb-domain–phosphate contacts are translocated by one nucleotide step, while the little-finger contacts with phosphate groups remain fixed. These stepwise conformational transitions accompanying nucleoside triphosphate binding and covalent nucleobase incorporation during a full replication cycle of Dpo4-catalyzed bypass of the oxoG lesion are distinct from the translocation events in replicative polymerases

Activities of human DNA polymerase κ 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 Polκ can effectively bypass a template (+)- trans- anti-BPDE- N 2 -dG adduct in an error-free manner. Kinetic parameters indicate that Polκ bypass of the (−)- trans- anti-BPDE- N 2 -dG adduct was ∼41-fold more efficient compared to the (+)- trans- anti-BPDE- N 2 -dG adduct. Furthermore, we have found another activity of human Polκ 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 Polβ and the purified catalytic subunits of yeast Polα, Polδ, and Polε right before the lesion. Extension by human Polκ 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 Polκ prior to DNA synthesis. Thus, sequence contexts 5′ to the lesion strongly affect the fidelity and mechanism of the Polκ-catalyzed extension synthesis. These results support a dual-function model of human Polκ 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

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

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 η (Polη) in mutagenesis induced by benzo[ a]pyrene DNA adducts in cells. Purified human Polη predominantly inserted an A opposite a template (+)- and (−)- trans- anti-BPDE- N 2-dG, two important DNA adducts of benzo[ a]pyrene. Both lesions also dramatically elevated G and T mis-insertion error rates of human Polη. Error-prone nucleotide insertion by human Polη was more efficient opposite the (+)- trans- anti-BPDE- N 2-dG adduct than opposite the (−)- trans- anti-BPDE- N 2-dG. However, translesion synthesis by human Polη largely stopped opposite the lesion and at one nucleotide downstream of the lesion (+1 extension). The limited extension synthesis of human Polη from opposite the lesion was strongly affected by the stereochemistry of the trans- anti-BPDE- N 2-dG adducts, the nucleotide opposite the lesion, and the sequence context 5′ to the lesion. By combining the nucleotide insertion activity of human Polη and the extension synthesis activity of human Polκ, effective error-prone lesion bypass was achieved in vitro in response to the (+)- and (−)- trans- anti-BPDE- N 2-dG DNA adducts

Implications for Damage Recognition during Dpo4-Mediated Mutagenic Bypass of m1G and m3C Lesions

DNA is susceptible to alkylation damage by a number of environmental agents that modify the Watson-Crick edge of the bases. Such lesions, if not repaired, may be bypassed by Y-family DNA polymerases. The bypass polymerase Dpo4 is strongly inhibited by 1-methylguanine (m1G) and 3-methylcytosine (m3C), with nucleotide incorporation opposite these lesions being predominantly mutagenic. Further, extension after insertion of both correct and incorrect bases, introduces additional base substitution and deletion errors. Crystal structures of the Dpo4 ternary extension complexes with correct and mismatched 3′-terminal primer bases opposite the lesions reveal that both m1G and m3C remain positioned within the DNA template/primer helix. However, both correct and incorrect pairing partners exhibit pronounced primer terminal nucleotide distortion, being primarily evicted from the DNA helix when opposite m1G or misaligned when pairing with m3C. Our studies provide insights into mechanisms related to hindered and mutagenic bypass of methylated lesions and models associated with damage recognition by repair demethylases.National Institutes of Health (U.S.) (Grant CA80024)United States. Dept. of Energy. Office of Basic Energy Sciences (DE-AC02-06CH11357)National Center for Research Resources (U.S.). Argonne National Laboratory (Award RR-15301

Response of human REV1 to different DNA damage: preferential dCMP insertion opposite the lesion

REV1 functions in the DNA polymerase ζ mutagenesis pathway. To help understand the role of REV1 in lesion bypass, we have examined activities of purified human REV1 opposite various template bases and several different DNA lesions. Lacking a 3′→5′ proofreading exonuclease activity, purified human REV1 exhibited a DNA polymerase activity on a repeating template G sequence, but catalyzed nucleotide insertion with 6-fold lower efficiency opposite a template A and 19–27-fold lower efficiency opposite a template T or C. Furthermore, dCMP insertion was greatly preferred regardless of the specific template base. Human REV1 inserted a dCMP efficiently opposite a template 8-oxoguanine, (+)-trans-anti-benzo[a]pyrene-N( 2)-dG, (–)-trans-anti-benzo[a]pyrene-N( 2)-dG and 1,N( 6)-ethenoadenine adducts, very inefficiently opposite an acetylaminofluorene-adducted guanine, but was unresponsive to a template TT dimer or TT (6–4) photoproduct. Surprisingly, the REV1 specificity of nucleotide insertion was very similar in response to different DNA lesions with greatly preferred C insertion and least frequent A insertion. By combining the dCMP insertion activity of human REV1 with the extension synthesis activity of human polymerase κ, bypass of the trans-anti-benzo[a]pyrene-N( 2) -dG adducts and the 1,N (6)-ethenoadenine lesion was achieved by the two-polymerase two-step mechanism. These results suggest that human REV1 is a specialized DNA polymerase that may contribute to dCMP insertion opposite many types of DNA damage during lesion bypass

Error-prone lesion bypass by human DNA polymerase η

DNA lesion bypass is an important cellular response to genomic damage during replication. Human DNA polymerase η (Polη), encoded by the Xeroderma pigmentosum variant ( XPV ) gene, is known for its activity of error-free translesion synthesis opposite a TT cis - syn cyclobutane dimer. Using purified human Polη, we have examined bypass activities of this polymerase opposite several other DNA lesions. Human Polη efficiently bypassed a template 8-oxoguanine, incorporating an A or a C opposite the lesion with similar efficiencies. Human Polη effectively bypassed a template abasic site, incorporating an A and less frequently a G opposite the lesion. Significant –1 deletion was also observed when the template base 5′ to the abasic site is a T. Human Polη partially bypassed a template (+)- trans - anti -benzo[ a ]pyrene- N 2 -dG and predominantly incorporated an A, less frequently a T, and least frequently a G or a C opposite the lesion. This specificity of nucleotide incorporation correlates well with the known mutation spectrum of (+)- trans - anti -benzo[a]pyrene- N 2 -dG lesion in mammalian cells. These results show that human Polη is capable of error-prone translesion DNA syntheses in vitro and suggest that Polη may bypass certain lesions with a mutagenic consequence in humans

Lesion bypass activities of human DNA polymerase mu

DNA polymerase mu (Polmu) is a newly discovered member of the polymerase X family with unknown cellular function. The understanding of Polmu function should be facilitated by an understanding of its biochemical activities. By using purified human Polmu for biochemical analyses, we discovered the lesion bypass activities of this polymerase in response to several types of DNA damage. When it encountered a template 8-oxoguanine, abasic site, or 1,N(6)-ethenoadenine, purified human Polmu efficiently bypassed the lesion. Even bulky DNA adducts such as N-2-acetylaminofluorene-adducted guanine, (+)- and (-)-trans-anti-benzo[a]pyrene-N(2)-dG were unable to block the polymerase activity of human Polmu. Bypass of these simple base damage and bulky adducts was predominantly achieved by human Polmu through a deletion mechanism. The Polmu specificity of nucleotide incorporation indicates that the deletion resulted from primer realignment before translesion synthesis. Purified human Polmu also effectively bypassed a template cis-syn TT dimer. However, this bypass was achieved in a mainly error-free manner with AA incorporation opposite the TT dimer. These results provide new insights into the biochemistry of human Polmu and show that efficient translesion synthesis activity is not strictly confined to the Y family polymerases