SITE-SPECIFICITY OF INCISIONS AT G-T AND O-6-METHYLGUANINE-T BASE MISMATCHES IN DNA BY HUMAN CELL-FREE-EXTRACTS

Cell-free extract from human tumor cell line A1235 (lacking O-6-methylguanine-DNA methyltransferase) was employed to compare incision at G:T base mispairs with that at O-6-methylguanine ((m6)G):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 (m6)G: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 bends 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. When the G:T mismatches were replaced by O-6-methylguanine:T ((m6)G:T) mismatches, the extract incised only DNAs with Cp(m6)G:T and Tp(m6)G:T pairs at both sites to nearly the same extent, but showed no detectable incision of DNAs having Ap(m6)G:T or Gp(m6)G:T pairs. Together, the results suggest that the human cell-free extract may process G:T base mismatches produced by spontaneous deamination of C-m5 and (m6)G:T pairs by more than one mechanism.