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Mutational analysis of the C8-guanine adduct of the environmental carcinogen 3-nitrobenzanthrone in human cells: critical roles of DNA polymerases ? and ? and Rev1 in error-prone translesion synthesis.

ABSTRACT: 3-Nitrobenzanthrone (3-NBA), a potent mutagen and suspected human carcinogen, is a common environmental pollutant. The genotoxicity of 3-NBA has been associated with its ability to form DNA adducts, including N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (C8-dG-ABA). To investigate the molecular mechanism of C8-dG-ABA mutagenesis in human cells, we have replicated a plasmid containing a single C8-dG-ABA in human embryonic kidney 293T (HEK293T) cells, which yielded 14% mutant progeny. The major types of mutations induced by C8-dG-ABA were G?T>G?A>G?C. siRNA knockdown of the translesion synthesis (TLS) DNA polymerases (pols) in HEK293T cells indicated that pol ?, pol ?, pol ?, pol ?, and Rev1 each have a role in replication across this adduct. The extent of TLS was reduced with each pol knockdown, but the largest decrease (of ?55% reduction) in the level of TLS occurred in cells with knockdown of pol ?. Pol ? and pol ? were considered the major contributors of the mutagenic TLS, because the mutation frequency (MF) decreased by 70%, when these pols were simultaneously knocked down. Rev1 also is important for mutagenesis, as reflected by the 60% reduction in MF upon Rev1 knockdown, but it probably plays a noncatalytic role by physically interacting with the other two Y-family pols. In contrast, pol ? appeared to be involved in the error-free bypass of the lesion, because MF increased by 60% in pol ? knockdown cells. These results provide important mechanistic insight into the bypass of the C8-dG-ABA adduct.

SUBMITTER: Pande P

PROVIDER: S-EPMC4139159 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Mutational analysis of the C8-guanine adduct of the environmental carcinogen 3-nitrobenzanthrone in human cells: critical roles of DNA polymerases η and κ and Rev1 in error-prone translesion synthesis.

Pande Paritosh P Malik Chanchal K CK Bose Arindam A Jasti Vijay P VP Basu Ashis K AK

Biochemistry 20140806 32

3-Nitrobenzanthrone (3-NBA), a potent mutagen and suspected human carcinogen, is a common environmental pollutant. The genotoxicity of 3-NBA has been associated with its ability to form DNA adducts, including N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (C8-dG-ABA). To investigate the molecular mechanism of C8-dG-ABA mutagenesis in human cells, we have replicated a plasmid containing a single C8-dG-ABA in human embryonic kidney 293T (HEK293T) cells, which yielded 14% mutant progeny. The major t ...[more]

PMID: 25080294

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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 ?, pol ?, pol ?, pol ?, or Rev1. Up to 75% reduction in TLS occurred when pol ?, pol ?, 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 ? resulted in an increase in MF, whereas MF was reduced when pol ?, pol ?, pol ?, or Rev1 was knocked down. The reduction in MF was most pronounced when pol ?, pol ?, 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 ? predominantly performs error-free TLS of the dG-N(2)-IQ adduct, whereas pols ?, pol ?, and Rev1 cooperatively carry out the error-prone TLS. However, in vitro experiments using yeast pol ? and ? 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.

Dataset Information

Mutational analysis of the C8-guanine adduct of the environmental carcinogen 3-nitrobenzanthrone in human cells: critical roles of DNA polymerases ? and ? and Rev1 in error-prone translesion synthesis.

Publications

Mutational analysis of the C8-guanine adduct of the environmental carcinogen 3-nitrobenzanthrone in human cells: critical roles of DNA polymerases η and κ and Rev1 in error-prone translesion synthesis.

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