title: Translesion Synthesis of the N(2)-2'-Deoxyguanosine Adduct of the Dietary Mutagen IQ in Human Cells: Error-Free Replication by DNA Polymerase κ and Mutagenic Bypass by DNA Polymerases η, ζ, and Rev1.
abstract: 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.
authors.affiliation: Department of Chemistry, University of Connecticut , Storrs, Connecticut 06269, United States.
authors.forename: Arindam
authors.initials: A
authors.lastname: Bose
authors.affiliation: Department of Chemistry, Vanderbilt University , Nashville, Tennessee 37232, United States.
authors.forename: Amy D
authors.initials: AD
authors.lastname: Millsap
authors.affiliation: Department of Chemistry, Vanderbilt University , Nashville, Tennessee 37232, United States.
authors.forename: Arnie
authors.initials: A
authors.lastname: DeLeon
authors.affiliation: Department of Chemistry, Vanderbilt University , Nashville, Tennessee 37232, United States.
authors.forename: Carmelo J
authors.initials: CJ
authors.lastname: Rizzo
authors.affiliation: Department of Chemistry, University of Connecticut , Storrs, Connecticut 06269, United States.
authors.forename: Ashis K
authors.initials: AK
authors.lastname: Basu
mesh_terms: D018736:DNA Adducts / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004261:DNA Replication / Q000187:drug effects; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003849:Deoxyguanosine / Q000096:biosynthesis; D004032:Diet; D057809:HEK293 Cells; D006801:Humans; D007093:Imidazoles / Q000633:toxicity; D007532:Isoleucine / Q000031:analogs & derivatives / Q000633:toxicity; D015394:Molecular Structure; D009153:Mutagens / Q000633:toxicity; D009687:Nuclear Proteins / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000378:metabolism*; D011810:Quinoxalines / Q000633:toxicity; D000094769:DNA Polymerase iota; D000099270:Y-Family DNA Polymerases
chemical_list: D018736:DNA Adducts; D004259:DNA-Directed DNA Polymerase; D003849:Deoxyguanosine; D007093:Imidazoles; D007532:Isoleucine; D009153:Mutagens; D009687:Nuclear Proteins; D009713:Nucleotidyltransferases; D011810:Quinoxalines; D000094769:DNA Polymerase iota; C516826:2-amino-3-methylpentanoic acid; C587420:imidazo(1,2-a)quinoxalin-4-amine; C429004:REV1 protein, human; D000099270:Y-Family DNA Polymerases; C000722070:POLI protein, human