Kinetics of bisulfite-induced cytosine deamination in single-stranded DNA.

Published

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Chen, H; Shaw, BR

Published Date

  • April 1993

Published In

Volume / Issue

  • 32 / 14

Start / End Page

  • 3535 - 3539

PubMed ID

  • 8466898

Pubmed Central ID

  • 8466898

Electronic International Standard Serial Number (EISSN)

  • 1520-4995

International Standard Serial Number (ISSN)

  • 0006-2960

Digital Object Identifier (DOI)

  • 10.1021/bi00065a003

Language

  • eng