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The abundant DNA adduct N 7-methyl deoxyguanosine contributes to miscoding during replication by human DNA polymerase ?.

ABSTRACT: Aside from abasic sites and ribonucleotides, the DNA adduct N 7-methyl deoxyguanosine (N7 -CH3 dG) is one of the most abundant lesions in mammalian DNA. Because N7 -CH3 dG is unstable, leading to deglycosylation and ring-opening, its miscoding potential is not well-understood. Here, we employed a 2'-fluoro isostere approach to synthesize an oligonucleotide containing an analog of this lesion (N7 -CH3 2'-F dG) and examined its miscoding potential with four Y-family translesion synthesis DNA polymerases (pols): human pol (hpol) ?, hpol ?, and hpol ? and Dpo4 from the archaeal thermophile Sulfolobus solfataricus We found that hpol ? and Dpo4 can bypass the N7 -CH3 2'-F dG adduct, albeit with some stalling, but hpol ? is strongly blocked at this lesion site, whereas hpol ? showed no distinction with the lesion and the control templates. hpol ? yielded the highest level of misincorporation opposite the adduct by inserting dATP or dTTP. Moreover, hpol ? did not extend well past an N 7-CH3 2'-F dG:dT mispair. MS-based sequence analysis confirmed that hpol ? catalyzes mainly error-free incorporation of dC, with misincorporation of dA and dG in 5-10% of products. We conclude that N 7-CH3 2'-F dG and, by inference, N 7-CH3 dG have miscoding and mutagenic potential. The level of misincorporation arising from this abundant adduct can be considered as potentially mutagenic as a highly miscoding but rare lesion.

SUBMITTER: Njuma OJ

PROVIDER: S-EPMC6664181 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Project description:Translesion synthesis (TLS) of the N(2)-2'-deoxyguanosine (dG-N(2)-IQ) adduct of the carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was investigated in human embryonic kidney 293T cells by replicating plasmid constructs in which the adduct was individually placed at each guanine (G1, G2, or G3) of the NarI sequence (5'-CG1G2CG3CC-3'). TLS efficiency was 38%, 29%, and 25% for the dG-N(2)-IQ located at G1, G2, and G3, respectively, which suggests that dG-N(2)-IQ is bypassed more efficiently by one or more DNA polymerases at G1 than at either G2 or G3. TLS efficiency was decreased 8-35% in cells with knockdown of pol η, pol κ, pol ι, pol ζ, or Rev1. Up to 75% reduction in TLS occurred when pol η, pol ζ, and Rev1 were simultaneously knocked down, suggesting that these three polymerases play important roles in dG-N(2)-IQ bypass. Mutation frequencies (MFs) of dG-N(2)-IQ at G1, G2, and G3 were 23%, 17%, and 11%, respectively, exhibiting a completely reverse trend of the previously reported MF of the C8-dG adduct of IQ (dG-C8-IQ), which is most mutagenic at G3 ( ( 2015 ) Nucleic Acids Res. 43 , 8340 - 8351 ). The major type of mutation induced by dG-N(2)-IQ was targeted G → T, as was reported for dG-C8-IQ. In each site, knockdown of pol κ resulted in an increase in MF, whereas MF was reduced when pol η, pol ι, pol ζ, or Rev1 was knocked down. The reduction in MF was most pronounced when pol η, pol ζ, and Rev1 were simultaneously knocked down and especially when the adduct was located at G3, where MF was reduced by 90%. We conclude that pol κ predominantly performs error-free TLS of the dG-N(2)-IQ adduct, whereas pols η, pol ζ, and Rev1 cooperatively carry out the error-prone TLS. However, in vitro experiments using yeast pol ζ and κ showed that the former was inefficient in full-length primer extension on dG-N(2)-IQ templates, whereas the latter was efficient in both error-free and error-prone extensions. We believe that the observed differences between the in vitro experiments using purified DNA polymerases, and the cellular results may arise from several factors including the crucial roles played by the accessory proteins in TLS.

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The abundant DNA adduct N 7-methyl deoxyguanosine contributes to miscoding during replication by human DNA polymerase ?.

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