Site-specific frame-shift mutagenesis by the 1-nitropyrene-DNA adduct N-(deoxyguanosin-8-y1)-1-aminopyrene located in the (CG)3 sequence: effects of SOS, proofreading, and mismatch repair.
1-Nitropyrene (1-NP), the predominant nitropolycyclic hydrocarbon found in diesel exhaust, is a mutagen and tumorigen. Nitroreduction is a major pathway by which 1-NP is metabolized. Reductively activated 1-NP forms a major DNA adduct, N-(deoxyguanosin-8-yl)-1-aminopyrene (dGAP), both in vitro and in vivo. In Salmonella typhimurium 1-NP induces a CpG deletion in a CGCGCGCG sequence. In Escherichia coli, however, mostly -1 and +1 frame-shifts are observed, which occur predominantly in 5'-CG, 5'-GC, and 5'-GG sequences. In order to determine the mechanism of mutagenesis by dGAP in a CpG repetitive sequence, we constructed a single-stranded M13 genome containing the adduct at the underscored deoxyguanosine of an inserted CGCGCG sequence. In E. coli strains with normal repair capability the adduct induced approximately 2% CpG deletions, which was 20-fold that of the control. With SOS, the frequency of frame-shift mutations increased to 2.6%, even though the frequency of CpG deletion accompanied 50% reduction. The enhancement in mutagenesis was due to a +1 frame-shift that occurred at a high frequency. In strains with a defect in methyl-directed mismatch repair, 50-70% increase in mutation frequency was observed. When these strains were SOS induced, frame-shift mutagenesis increased by approximately 100%. When transfections were carried out in dnaQ strains that are impaired in 3'-->5'exonuclease activity of DNA polymerase III, frame-shift mutagenesis increased 5-7-fold. dGAP-induced frame-shifts in the (CG)3 sequence, therefore, varied from 2% to 17% depending on the state of repair of the host cells. We conclude that dGAP induces both -2 and +1 frame-shifts in a CpG repetitive sequence and that these two mutagenic events are competing pathways. The CpG deletion does not require SOS functions, whereas the +1 frame-shifts are SOS-dependent. On the basis of the data in repair-deficient strains, it appears that both types of frame-shifts occurred as a result of misalignment, which are corrected primarily by the proofreading exonuclease of the DNA polymerase. Misaligned structures that escape the exonuclease are repaired by the methyl-directed mismatch repair, albeit with limited efficiency.
Malia
S A
SA
Department of Chemistry, University of Connecticut, Storrs 06269, USA.
Vyas
R R
RR
Basu
A K
AK
D017104:Bacteriophage M13 / Q000235:genetics; D001483:Base Sequence; D018736:DNA Adducts / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D004260:DNA Repair; D004269:DNA, Bacterial / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D016368:Frameshift Mutation; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009153:Mutagens / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity*; D011721:Pyrenes / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity*; D012329:RNA, Bacterial / Q000235:genetics; D013014:SOS Response, Genetics; D012486:Salmonella typhimurium / Q000187:drug effects / Q000235:genetics / Q000378:metabolism
D018736:DNA Adducts; D004269:DNA, Bacterial; D009153:Mutagens; D011721:Pyrenes; D012329:RNA, Bacterial; C045229:N-(deoxyguanosin-8-yl)-1-aminopyrene; D003849:Deoxyguanosine; C032668:1-nitropyrene