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 mispairs in DNA have been assayed on duplex substrates containing modified guanine bases. The first, the G.T mismatch incision activity, is specifically involved in short-patch repair of mispairs arising via deamination of 5-methylcytosine. The second activity can be detected by its ability td bind to G.T mispairs and may initiate correction by a long-patch 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 was paired with cytosine. Extracts of cells that are tolerant both to methylation damage and to 6-thioguanine in DNA also incised 6-thioguanine T and O-6-methylguanine.T base pairs. The data suggest that this activity is unlikely to contribute significantly to the biological effects 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 were recognized efficiently and to an equivalent extent by the same protein complex as G.T mispairs. O-6-Methylguanine T base pairs were also recognized but with reduced efficiency. No binding was observed to 6-thioguanine C or O-6-methylguanine C base pairs. Recognition by the binding complex was essentially independent of the base immediately 5' to the mismatched guanine but was somewhat more efficient if O-6-methylguanine was preceded by a purine. Extracts of two tolerant lines with a known defect in G.T mismatch binding failed to form complexes with substrates 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 involved in mediating the cytotoxicity of the two-base analogs.