INFLUENCE OF DNA-SEQUENCE IDENTITY ON EFFICIENCY OF TARGETED GENE REPLACEMENT

We have developed a system for analyzing recombination between a DNA f ragment released in the nucleus from a single-copy plasmid and a genom ic target in order to determine the influence of DNA sequence mismatch es on the frequency of gene replacement in Saccharomyces cerevisiae. M ismatching was shown to be a potent barrier to efficient gene replacem ent, but its effect was considerably ameliorated by the presence of DN A sequences that are identical to the genomic target at one end of a c himeric DNA fragment, Disruption of the mismatch repair gene MSH2 grea tly reduces but does not eliminate the barrier to recombination betwee n mismatched DNA fragment and genomic target sequences, indicating tha t the inhibition of gene replacement with mismatched sequences is at l east partially under the control of mismatch repair. We also found tha t mismatched sequences inhibited recombination between a DNA fragment and the genome only when they were close to the edge of the fragment. Together these data indicate that while mismatches can destabilize the relationship between a DNA fragment and a genomic target sequence, th ey will only do so if they are likely to be in the heteroduplex formed between the recombining molecules.