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.
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.
authors: Department of Chemistry, University of Connecticut , Storrs, Connecticut 06269, United States.
authors: Paritosh
authors: P
authors: Pande
authors: Chanchal K
authors: CK
authors: Malik
authors: Arindam
authors: A
authors: Bose
authors: Vijay P
authors: VP
authors: Jasti
authors: Ashis K
authors: AK
authors: Basu
mesh_terms: D001551:Benz(a)Anthracenes / Q000633:toxicity*; D002274:Carcinogens, Environmental / Q000633:toxicity; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000633:toxicity; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D015972:Gene Expression Regulation, Neoplastic; D057809:HEK293 Cells; D006801:Humans; D015394:Molecular Structure; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering; D000099270:Y-Family DNA Polymerases
chemical_list: D001551:Benz(a)Anthracenes; D002274:Carcinogens, Environmental; D004259:DNA-Directed DNA Polymerase; D003849:Deoxyguanosine; D009687:Nuclear Proteins; D009713:Nucleotidyltransferases; D034741:RNA, Small Interfering; C000622103:N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone; C429004:REV1 protein, human; D000099270:Y-Family DNA Polymerases; C404302:POLK protein, human; C112338:Rad30 protein; C117220:3-nitrobenzanthrone