Kinetics of bisulfite-induced cytosine deamination in single-stranded DNA.
The rate of bisulfite-induced deamination of cytosine to uracil in single-stranded (ss) DNA at physiological temperature and pH was monitored by a sensitive genetic assay. The assay is based on reversion of a mutation in the lacZ alpha gene of bacteriophage M13mp2 and employs ung- (NR9404) and ung+ (MC1061) bacterial strains which are isogenic except for uracil glycosylase activity. For ss DNA incubated with 1-50 mM bisulfite and transfected into an ung- cell strain, the reversion frequency increased linearly with time of incubation and with concentration of bisulfite. Of 54 revertants sequenced, all were C-->T transitions. Reduction in reversion frequency upon transfecting ss DNA into ung+ cells indicated that the majority of mutations were occurring via a uracil intermediate. Assuming that all revertants arose via uracil, the pseudo-first-order rate constant for deamination in 10 mM sodium bisulfite and 10 mM Hepes-NaOH, pH 7.4, at 37 degrees C as measured by transfecting into an ung- cell strain was 3.5 x 10(-10) s-1, as compared to a spontaneous background rate constant of 0.6 x 10(-10) s-1 in buffer alone.
Chen
H
H
Department of Chemistry, Duke University, Durham, North Carolina 27708-0346.
Shaw
B R
BR
D001435:Bacteriophages / Q000235:genetics; D003062:Codon; D003596:Cytosine / Q000378:metabolism*; D045647:DNA Glycosylases; D004260:DNA Repair / Q000235:genetics; D004277:DNA, Single-Stranded / Q000187:drug effects / Q000378:metabolism*; D003641:Deamination; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000378:metabolism; D013447:Sulfites / Q000494:pharmacology*; D014169:Transformation, Bacterial; D014498:Uracil / Q000378:metabolism; D051981:Uracil-DNA Glycosidase; D001616:beta-Galactosidase / Q000235:genetics
D003062:Codon; D004277:DNA, Single-Stranded; D013447:Sulfites; D014498:Uracil; D003596:Cytosine; D001616:beta-Galactosidase; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase; C009279:sodium bisulfite