Title

Mechanism of mutagenesis by the 1-nitropyrene-DNA adduct {\it N\/}-(deoxyguanosin-8-yl)-1-aminopyrene

Date of Completion

January 1996

Keywords

Biology, Molecular|Health Sciences, Toxicology|Chemistry, Biochemistry

Degree

Ph.D.

Abstract

This study investigated the mutational specificity of the major DNA adduct formed by reductively activated 1-nitropyrene, N-(deoxyguanosin-8-yl)-1-aminopyrene (dG$\rm\sp{AP}$), in Escherichia coli. The distribution of DNA adducts was studied in single-stranded M13mp18 DNA. Replication inhibition, primarily at or 3$\sp\prime$ to guanine bases, was observed. Transfection of the modified M13 DNA into E. coli cells indicated a dose-dependent reduction in viability with concomitant enhancement in mutagenesis in the lacZ gene fragment. With SOS induction, one-base deletions and insertions were the major event (45%), although base substitutions also occurred at high frequency (44%). A major proportion of the point mutations, and particularly one-base deletions and insertions, were detected in 5$\sp\prime$-CG, 5$\sp\prime$-GC, or 5$\sp\prime$-GG sequences. Without SOS functions, the major type of mutation was the C$\to$T transition (48%). Further studies have shown that cytosine deamination occurred during ascorbic acid-induced nitroreduction, which was likely responsible for these mutations.^ Site-specific mutagenesis assays were conducted with an M13 ss genome containing a single dG$\rm\sp{AP}$ lesion 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 induced, the frequency of frame-shift mutations increased to 2.6%, even though the frequency of CpG deletion accompanied a 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, a 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$\sp\prime$-5$\sp\prime$ exonuclease activity of DNA polymerase III, frame-shift mutagenesis increased 5-7 fold. It is concluded from these studies that dG$\rm\sp{AP}$ induced both $-$2 and +1 frame-shifts in a CpG repetitive sequence. 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. ^