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Title: 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.

Authors: Pande, Paritosh; Malik, Chanchal K; Bose, Arindam; Jasti, Vijay P; Basu, Ashis K

Published In Biochemistry, (2014 Aug 19)

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 η, pol κ, pol ι, pol ζ, 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 ζ. Pol η and pol κ 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 ζ appeared to be involved in the error-free bypass of the lesion, because MF increased by 60% in pol ζ knockdown cells. These results provide important mechanistic insight into the bypass of the C8-dG-ABA adduct.

PubMed ID: 25080294 Exiting the NIEHS site

MeSH Terms: Benz(a)Anthracenes/toxicity*; Carcinogens, Environmental/toxicity; DNA-Directed DNA Polymerase/chemistry; DNA-Directed DNA Polymerase/metabolism*; Deoxyguanosine/analogs & derivatives*; Deoxyguanosine/toxicity; Gene Expression Regulation, Enzymologic/physiology; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Molecular Structure; Mutation; Nuclear Proteins/genetics; Nuclear Proteins/metabolism*; Nucleotidyltransferases/genetics; Nucleotidyltransferases/metabolism*; RNA Interference; RNA, Small Interfering

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