DIFFERENT REPAIR KINETICS FOR SHORT AND LONG DNA DOUBLE-STRAND GAPS IN SACCHAROMYCES-CEREVISIAE

The kinetics of recombinational repair of plasmid DNA double-strand br eaks (dsb) and gaps (dsg) of differnt sizes and ends were studied. For this purpose we used the mutant rad54-3 of the yeast Saccharomyces ce revisiae, which is temperature dependent with respect to genetic recom bination and rejoining of dsb/dsg; allowing us to stop these processes by shifting cells to the restrictive temperature. We found that the k inetics of repair of cohesive-ended dsb and small gaps (up to 400 bp) are similar and characterized by two phases separated by a plateau. In contrast, large gap (1.4 kbp) repair proceeds with different kinetics exhibiting only the second phase. We also investigated the repair kin etics of 400 bp gaps introduced into plasmid DNA with and without homo logy to chromosomal DNA allowing recombinational repair and non-recomb inational repair (ligation), respectively. We found that gaps introduc ed in plasmid sequences homologous to chromosomal DNA are rapidly repa ired by recombination. In contrast, recircularization of the gapped pl asmid by ligation is as slow and inefficient as ligation of a cohesive -ended dsb. The kinetics of repair of gapped plasmids may be explained by assuming a constitutive level of enzymes responsible for the first phase of recombinational repair, while inducible enzymes, which becom e available at the end of the plateau, carry out the second phase of r epair.