SITE-SPECIFIC FRAME-SHIFT MUTAGENESIS BY THE 1-NITROPYRENE-DNA ADDUCTN-(DEOXYGUANOSIN-8-Y1)-1-AMINOPYRENE LOCATED IN THE (CG)(3) SEQUENCE - EFFECTS OF SOS, PROOFREADING, AND MISMATCH REPAIR
Sa. Malia et al., SITE-SPECIFIC FRAME-SHIFT MUTAGENESIS BY THE 1-NITROPYRENE-DNA ADDUCTN-(DEOXYGUANOSIN-8-Y1)-1-AMINOPYRENE LOCATED IN THE (CG)(3) SEQUENCE - EFFECTS OF SOS, PROOFREADING, AND MISMATCH REPAIR, Biochemistry, 35(14), 1996, pp. 4568-4577
1-Nitropyrene (1-NP), the predominant nitropolycyclic hydrocarbon foun
d in diesel exhaust, is a mutagen and tumorigen. Nitroreduction is a m
ajor pathway by which 1-NP is metabolized. Reductively activated 1-NP
forms a major DNA adduct, N-(deoxyguanosin-8-yl)-1-aminopyrene (dG(AP)
), both in vitro and in vivo. In Salmonella typhimurium 1-NP induces a
CPG deletion in a CGCGCGCG sequence. In Escherichia coli, however, mo
stly -1 and +1 frame-shifts are observed, which occur predominantly in
5'-CG, 5'-GC, and 5'-GG sequences. In order to determine the mechanis
m of mutagenesis by dG(AP) in a CpG repetitive sequence, we constructe
d a single-stranded M13 genome containing the adduct at the underscore
d deoxyguanosine of an inserted CGCGCG-sequence. In E. coli strains wi
th normal repair capability the adduct induced approximately 2% CpG de
letions, which was 20-fold that of the control. With SOS, the frequenc
y of frame-shift mutations increased to 2.6%, even though the frequenc
y of CpG deletion accompanied 50% reduction. The enhancement in mutage
nesis was due to a +1 frame-shift that occurred at a high frequency. I
n strains with a defect in methyl-directed mismatch repair, 50-70% inc
rease 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 mutagen
esis increased 5-7-fold. dG(AP)-induced frame-shifts in the (CG)(3) se
quence, therefore, varied from 2% to 17% depending on the state of rep
air of the host cells. We conclude that dG(AP) 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 o
f frame-shifts occurred as a result of misalignment, which are correct
ed primarily by the proofreading exonuclease of the DNA polymerase. Mi
saligned structures that escape the exonuclease are repaired by the me
thyl-directed mismatch repair, albeit with limited efficiency.