Deamination of single-stranded DNA cytosine residues in aerobic nitric oxide solution at micromolar total NO exposures

Deamination of cytosine to uracil is a potential source of mutations in DNA. Here we examine the deaminating ability of aerobic nitric oxide (NO) toward single-stranded DNA at very low (micromolar and below) total exposures, using a sensitive genetic method that allows us to study a single deamination event at a specific site in a 7200-nucleotide DNA molecule within a pool of ca. 100 000 other identical DNA molecules. We incubated gapped C141 M13mp2 DNA with the NO-generating compound, Et(2)N[N(O)NO]Na (DEA/NO), in aerobic buffer for 16 h to ensure complete autoxidation at pH 7.4 and 37 degrees C. After ultrafiltration to remove small molecules, the DNA was transformed into isogenic Escherichia coli cultures that were either deficient (NR9404, ung(-)) or proficient (MC1061, ung(+)) in uracil-DNA glycosylase activity. The gapped DNA was constructed such that the target (CCC) codon was contained in a short single-stranded segment of otherwise double-stranded circular DNA, and the incubation was performed in a closed system to prevent loss of NO to the atmosphere before the reaction was complete. An increase in the reversion frequency in the ung(-) strain was noted between 0 and 1 mu M DEA/NO, and the reversion frequency leveled out between 3 and 30 mu M. However, 30 mu M ''spent'' DEA/NO (i.e., that which was similarly incubated for 4 h to complete the autoxidation of NO before the DNA was added) did not increase reversion frequency relative to control. Nearly all (42/43) of the mutations identified after 1 mu M DEA/NO treatment were C --> T transitions, and reversion frequency in the isogenic ung(+) strain was lower than in the ung(-) strain. The data are consistent with the hypothesis that total NO exposures in the mu mol/L range can lead to C --> T mutations via a mechanism most probably involving deamination of DNA cytosine residues.