Dataset Information

Formation of S-[2-(N6-Deoxyadenosinyl)ethyl]glutathione in DNA and Replication Past the Adduct by Translesion DNA Polymerases.

ABSTRACT: 1,2-Dibromoethane (DBE, ethylene dibromide) is a potent carcinogen due at least in part to its DNA cross-linking effects. DBE cross-links glutathione (GSH) to DNA, notably to sites on 2'-deoxyadenosine and 2'-deoxyguanosine ( Cmarik , J. L. , et al. ( 1991 ) J. Biol. Chem. 267 , 6672 - 6679 ). Adduction at the N6 position of 2'-deoxyadenosine (dA) had not been detected, but this is a site for the linkage of O6-alkylguanine DNA alkyltransferase ( Chowdhury , G. , et al. ( 2013 ) Angew. Chem. Int. Ed. 52 , 12879 - 12882 ). We identified and quantified a new adduct, S-[2-(N6-deoxyadenosinyl)ethyl]GSH, in calf thymus DNA using LC-MS/MS. Replication studies were performed in duplex oligonucleotides containing this adduct with human DNA polymerases (hPols) ?, ?, and ?, as well as with Sulfolobus solfataricus Dpo4, Escherichia coli polymerase I Klenow fragment, and bacteriophage T7 polymerase. hPols ? and ?, Dpo4, and Klenow fragment were able to bypass the adduct with only slight impedance; hPol ? and ? showed increased misincorporation opposite the adduct compared to that of unmodified 2'-deoxyadenosine. LC-MS/MS analysis of full-length primer extension products by hPol ? confirmed the incorporation of dC opposite S-[2-(N6-deoxyadenosinyl)ethyl]GSH and also showed the production of a -1 frameshift. These results reveal the significance of N6-dA GSH-DBE adducts in blocking replication, as well as producing mutations, by human translesion synthesis DNA polymerases.

SUBMITTER: Sedgeman CA

PROVIDER: S-EPMC5768145 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Formation of S-[2-(N<sup>6</sup>-Deoxyadenosinyl)ethyl]glutathione in DNA and Replication Past the Adduct by Translesion DNA Polymerases.

Sedgeman Carl A CA Su Yan Y Guengerich F Peter FP

Chemical research in toxicology 20170414 5

1,2-Dibromoethane (DBE, ethylene dibromide) is a potent carcinogen due at least in part to its DNA cross-linking effects. DBE cross-links glutathione (GSH) to DNA, notably to sites on 2'-deoxyadenosine and 2'-deoxyguanosine ( Cmarik , J. L. , et al. ( 1991 ) J. Biol. Chem. 267 , 6672 - 6679 ). Adduction at the N6 position of 2'-deoxyadenosine (dA) had not been detected, but this is a site for the linkage of O<sup>6</sup>-alkylguanine DNA alkyltransferase ( Chowdhury , G. , et al. ( 2013 ) Angew. ...[more]

PMID: 28395138

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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

Formation of S-[2-(N6-Deoxyadenosinyl)ethyl]glutathione in DNA and Replication Past the Adduct by Translesion DNA Polymerases.

Publications

Formation of S-[2-(N<sup>6</sup>-Deoxyadenosinyl)ethyl]glutathione in DNA and Replication Past the Adduct by Translesion DNA Polymerases.

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