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 efficient gene conversion process initiated by a double-strand break. The use of a galactose-inducible HO endonuclease gene has made it possible to analyze the synchronous progression of molecular intermediates during recombination. When MATa switches to MAT alpha, a 3' single-stranded end of HO-cleaved MAT DNA invades the homologous donor, HML alpha, and initiates copying of new DNA sequences. These early steps of recombination can be detected by PCR amplification. When recombination is initiated in a strain carrying the MATa-stk T-->A base pair substitution mutation located 8 bp to the right of the HO endonuclease cleavage site, the stk mutation is frequently included in heteroduplex DNA formed between MAT and HML and undergoes mismatch correction. We have followed the kinetics of mismatch repair of the stk mutation by determining the DNA sequence of the PCR-amplified early intermediates of recombination. Mismatch correction of heteroduplex DNA is quite rapid (t1/2 = 6-10 min) compared to the 60 min required to complete repair of the double-strand break. Mismatch repair occurs soon after the 3'-ended MAT-stk strand invades HML and forms heteroduplex DNA. Moreover, nearly all the correction events are restorations, in which the invading MAT-stk strand is corrected to the genotype of the resident HML donor. This rapid restoration ensures that the net result will be a gene conversion at the MAT locus. Rapid and preferential mismatch repair of heteroduplex DNA has important implications in understanding meiotic recombination.
Haber
J E
JE
Rosenstiel Center, Brandeis University, Waltham, MA 02254.
Ray
B L
BL
Kolb
J M
JM
White
C I
CI
D001483:Base Sequence; D004260:DNA Repair; D004271:DNA, Fungal / Q000235:genetics* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D005800:Genes, Fungal; D049770:Genes, Mating Type, Fungal; D000072235:Mating Factor; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics* / Q000378:metabolism; D010455:Peptides / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins
D004271:DNA, Fungal; D009692:Nucleic Acid Heteroduplexes; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; D000072235:Mating Factor; C530845:HO protein, S cerevisiae; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific