DNA MISMATCH BINDING AND INCISION AT MODIFIED GUANINE BASES BY EXTRACTS OF MAMMALIAN-CELLS - IMPLICATIONS FOR TOLERANCE TO DNA METHYLATION DAMAGE

Two activities involved in separate pathways for correcting G.T mispai rs in DNA have been assayed on duplex substrates containing modified g uanine bases. The first, the G.T mismatch incision activity, is specif ically involved in short-patch repair of mispairs arising via deaminat ion of 5-methylcytosine. The second activity can be detected by its ab ility td bind to G.T mispairs and may initiate correction by a long-pa tch mechanism. 6-Thioguanine and O-6-methylguanine paired with thymine were efficiently incised by cell extracts if the modified guanine was in a CpG dinucleotide. Incision was not observed when either purine w as paired with cytosine. Extracts of cells that are tolerant both to m ethylation damage and to 6-thioguanine in DNA also incised 6-thioguani ne T and O-6-methylguanine.T base pairs. The data suggest that this ac tivity is unlikely to contribute significantly to the biological effec ts of O-6-methylguanine in DNA. A defect in this pathway is therefore unlikely to explain the cross-resistance of tolerant cells to the two base analogs in DNA. In binding assays, 6-thioguanine T base pairs wer e recognized efficiently and to an equivalent extent by the same prote in complex as G.T mispairs. O-6-Methylguanine T base pairs were also r ecognized but with reduced efficiency. No binding was observed to 6-th ioguanine C or O-6-methylguanine C base pairs. Recognition by the bind ing complex was essentially independent of the base immediately 5' to the mismatched guanine but was somewhat more efficient if O-6-methylgu anine was preceded by a purine. Extracts of two tolerant lines with a known defect in G.T mismatch binding failed to form complexes with sub strates containing the modified bases. The ability of the G.T binding factor to recognize both O-6-methylguanine T and 6-thioguanine T pairs indicates that the long-patch repair pathway is more likely to be inv olved in mediating the cytotoxicity of the two-base analogs.