Bisulfite-induced cytosine deamination rates in E. coli SSB:DNA complexes.
E. coli single-stranded binding protein (SSB) has been examined for its ability to modulate bisulfite-induced cytosine deamination rates in single-stranded DNA (ssDNA). We used a lacZ alpha-complementation reversion assay to detect C-->U rates at a single codon in M13mp2 DNA, whether in free ssDNA or in an SSB:ssDNA complex. When incubated at 37 degrees C, the average bisulfite-induced reversion rate constant was four-fold less in SSB:ssDNA complexes than in ssDNA, at a single codon. Across a 250 base pair target and over 23 scorable C-->U sites, the forward rate constant was 4.9-fold less in SSB:ssDNA complexes than in ssDNA alone. After treatment with N-uracil glycosylase, ssDNA incubated with bisulfite had reversion frequencies at the background rate of ssDNA incubated without bisulfite, indicating that virtually all mutations scored were due to C-->U events. The decrease in cytosine deamination rates occurred both in a single codon and over a 250 bp target, indicating that interactions between SSB and ssDNA reduce bisulfite-catalyzed mutations. The structural role of SSB is well recognized in multiple cellular processes; SSB can also function to minimize bisulfite-induced ssDNA mutations.
Lough
J
J
PRA International, 100 Carolyn Circle, Bristol, TN 37620, USA.
Jackson
M
M
Morris
R
R
Moyer
R
R
D003596:Cytosine / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000378:metabolism* / Q000494:pharmacology; D003641:Deamination / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D005787:Gene Frequency; D046911:Macromolecular Substances; D009154:Mutation; D013447:Sulfites / Q000494:pharmacology*
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D013447:Sulfites; D003596:Cytosine; C042345:hydrogen sulfite