A sensitive genetic assay for the detection of cytosine deamination: determination of rate constants and the activation energy.
Previously it has not been possible to determine the rate of deamination of cytosine in DNA at 37 degrees C because this reaction occurs so slowly. We describe here a sensitive genetic assay to measure the rate of cytosine deamination in DNA at a single cytosine residue. The assay is based on reversion of a mutant in the lacZ alpha gene coding sequence of bacteriophage M13mp2 and employs ung- bacterial strains lacking the enzyme uracil glycosylase. The assay is sufficiently sensitive to allow us to detect, at a given site, a single deamination event occurring with a background frequency as low as 1 in 200,000. With this assay, we determined cytosine deamination rate constants in single-stranded DNA at temperatures ranging from 30 to 90 degrees C and then calculated that the activation energy for cytosine deamination in single-stranded DNA is 28 +/- 1 kcal/mol. At 80 degrees C, deamination rate constants at six sites varied by less than a factor of 3. At 37 degrees C, the cytosine deamination rate constants for single- and double-stranded DNA at pH 7.4 are 1 x 10(-10) and about 7 x 10(-13) per second, respectively. (In other words, the measured half-life for cytosine in single-stranded DNA at 37 degrees C is ca. 200 years, while in double-stranded DNA it is on the order of 30,000 years.) Thus, cytosine is deaminated approximately 140-fold more slowly when present in the double helix. These and other data indicate that the rate of deamination is strongly dependent upon DNA structure and the degree of protonation of the cytosine. The data suggest that agents which perturb DNA structure or facilitate direct protonation of cytosine may induce deamination at biologically significant rates. The assay provides a means to directly test the hypothesis.
Department of Chemistry, Duke University, Durham, North Carolina 27706.
L A
LA
Frederico
T A
TA
Kunkel
B R
BR
Shaw
D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D003596:Cytosine / Q000378:metabolism*; D045647:DNA Glycosylases; D004279:DNA, Viral / Q000235:genetics*; D003641:Deamination; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D009699:N-Glycosyl Hydrolases; D009710:Nucleotide Mapping; D013696:Temperature; D013816:Thermodynamics; D014162:Transfection; D051981:Uracil-DNA Glycosidase
D004279:DNA, Viral; D003596:Cytosine; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase