RAPID KINETICS OF MISMATCH REPAIR OF HETERODUPLEX DNA THAT IS FORMED DURING RECOMBINATION IN YEAST

Homothallic switching of yeast mating type (MAT) genes is a highly eff icient gene conversion process initiated by a double-strand break. The use of a galactose-inducible HO endonuclease gene has made it possibl e to analyze the synchronous progression of molecular intermediates du ring recombination. When MATa switches to MATalpha, a 3' single-strand ed end of HO-cleaved MAT DNA invades the homologous donor, HMLalpha, a nd initiates copying of new DNA sequences. These early steps of recomb ination can be detected by PCR amplification. When recombination is in itiated in a strain carrying the MATa-stk T --> A base pair substituti on mutation located 8 bp to the right of the HO endonuclease cleavage site, the stk mutation is frequently included in heteroduplex DNA form ed between MAT and HML and undergoes mismatch correction. We have foll owed the kinetics of mismatch repair of the stk mutation by determinin g the DNA sequence of the PCR-amplified early intermediates of recombi nation. Mismatch correction of heteroduplex DNA is quite rapid (t1/1 = 6-10 min) compared to the 60 min required to complete repair of the d ouble-strand break. Mismatch repair occurs soon after the 3'-ended MAT -stk strand invades HML and forms heteroduplex DNA. Moreover, nearly a ll the correction events are restorations, in which the invading MA T- stk strand is corrected to the genotype of the resident HML donor. Thi s rapid restoration ensures that the net result will be a gene convers ion at the MAT locus. Rapid and preferential mismatch repair of hetero duplex DNA has important implications in understanding meiotic recombi nation.