Site specificity of incisions at G:T and O6-methylguanine:T base mismatches in DNA by human cell-free extracts.
Cell-free extract from human tumor cell line A1235 (lacking O6-methylguanine-DNA methyltransferase) was employed to compare incision at G:T base mispairs with that at O6-methylguanine (m6G):T pairs at two different sites (sites 20 and 25) in 45-bp heteroduplexes. To study the effect of neighboring bases on the activity(ies), the base pair immediately 5' to the mismatched G at each site was varied to provide four contexts: CpG:T, TpG:T, ApG:T, and GpG:T (and two analogous series for m6G:T pairs). At site 20, cell-free extract produced observable incision only in the 45-bp DNA with the G:T mispair in the CpG:T context, giving a product with incisions immediately 5' and 3' to the mismatched T. We observed incision of neither the strand containing the mismatched G nor the DNAs with the site 20 ApG:T, GpG:T, and TpG:T mismatches. By contrast, at site 25, incision specificity was different. All four G:T mismatched DNAs were incised, and the ApG:T-25, GpG:T-25, and TpG:T-25 DNAs were incised 1-3 bonds 3' to the mismatched T, while similar in other respects to the CpG:T-25 DNA, which showed a pattern like the CpG:T-20 DNA. CpG:T-20 specific incision activity in the extract was strongly inhibited by both CpG:T (sites 20 and 25) DNAs, but at least 10-fold more poorly by DNAs with Apg:T-25 and GpG:T-25 pairs.(ABSTRACT TRUNCATED AT 250 WORDS)
Sibghat-Ullah
Department of Medicine, Cross Cancer Institute, Edmonton, Alberta, Canada.
Day
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D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002474:Cell-Free System; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D013941:Thymine / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D006147:Guanine; D004247:DNA; C008449:O-(6)-methylguanine; D013941:Thymine