INDUCTION OF RECOMBINATION BETWEEN HOMOLOGOUS AND DIVERGED DNAS BY DOUBLE-STRAND GAPS AND BREAKS AND ROLE OF MISMATCH REPAIR

Sequence homology is expected to influence recombination. To further u nderstand mechanisms of recombination and the impact of reduced homolo gy, we examined recombination during transformation between plasmid-bo rne DNA flanking a double-strand break (DSB) or gap and its chromosoma l homolog. Previous reports have concentrated on spontaneous recombina tion or initiation by undefined lesions. Sequence divergence of approx imately 16% reduced transformation frequencies by at least 10-fold. Ge ne conversion patterns associated with double-strand gap repair of epi somal plasmids or with plasmid integration were analyzed by restrictio n endonuclease mapping and DNA sequencing. For episomal plasmids carry ing homeologous DNA, at least one input end was always preserved beyon d 10 bp, whereas for plasmids carrying homologous DNA, both input ends were converted beyond 80 bp in 60% of the transformants. The system a llowed the recovery of transformants carrying mixtures of recombinant molecules that might arise if heteroduplex DNA-a presumed recombinatio n intermediate-escapes mismatch repair. Gene conversion involving homo logous DNAs frequently involved DNA mismatch repair, directed to a bro ken strand. A mutation in the PMS1 mismatch repair gene significantly increased the fraction of transformants carrying a mixture of plasmids for homologous DNAs, indicating that PMS1 can participate in DSB-init iated recombination. Since nearly all transformants involving homeolog ous DNAs carried a single recombinant plasmid in both Pms(+) and Pms(- ) strains, stable heteroduplex DNA appears Less likely than for homolo gous DNAs. Regardless of homology, gene conversion does not appear to occur by nucleolytic expansion of a DSB to a gap prior to recombinatio n. The results with homeologous DNAs are consistent with a recombinati onal repair model that we propose does not require the formation of st able heteroduplex DNA but instead involves other homology-dependent in teractions that allow recombination-dependent DNA synthesis.