GENE TARGETING BY LINEAR DUPLEX DNA FREQUENTLY OCCURS BY ASSIMILATIONOF A SINGLE-STRAND THAT IS SUBJECT TO PREFERENTIAL MISMATCH CORRECTION

To study targeted recombination, a single linear 2-kb fragment of LEU2 DNA was liberated from a chromosomal site within the nucleus of Sacch aromyces cerevisiae, by expression of the site-specific HO endonucleas e, Gene targeting was scored by gene conversion of a chromosomal leu2 mutant allele by the liberated LEU2 fragment, This occurred at a frequ ency of only 2 x 10(-4), despite the fact that nearly all cells succes sfully repaired, by single-strand annealing, the chromosome break crea ted by liberating the fragment, The frequency of Leu(+) recombinants w as 6- to 25-fold higher in pms1 strains lacking mismatch repair, In 70 % of these cases, the colony was sectored for Leu(+)/Leu(-). Similar r esults were obtained when a 4.1-kb fragment containing adjacent LEU2 a nd ADE1 genes was liberated, to convert adjacent leu2 and ade1 mutatio ns on the chromosome, These results suggest that a linear fragment is not assimilated into the recipient chromosome by two crossovers each c lose to the end of the fragment; rather, heteroduplex DNA between the fragment and the chromosome is apparently formed over the entire regio n, by the assimilation of one of the two strands of the linear duplex DNA, Moreover, the recovery of Leu(+) transformants is frequently defe ated by the cell's mismatch repair machinery; more than 85% of mismatc hes in heteroduplex DNA are corrected in favor of the resident, unbrok en (mutant) strand.