PULSED-FIELD GEL ANALYSIS OF THE PATTERN OF DNA DOUBLE-STRAND BREAKS IN THE SACCHAROMYCES GENOME DURING MEIOSIS

Pulsed-field gel electrophoresis (PFGE) has been used to study the tim ing, frequency, and distribution of double-strand breaks (DSBs) in chr omosomal-sized DNA during meiosis in yeast. It has previously been sho wn that DSBs are associated with some genetic hotspots during recombin ation, and it is important to know whether meiotic recombination event s routinely initiate via DSBs. Two strains have been studied here-a hi gh-sporulating homothallic wild type and a congenic mutant strain carr ying a rad50S mutation. This mutant has previously been reported to ac cumulate broken molecules in meiosis to much higher frequencies than w ild type and to abolish the characteristic wild-type processing of DNA that has been observed at the break sites. When whole chromosomes are resolved by PFGE, both strains show some broken molecules starting at the time that cells commit to genetic recombination. Breakage has bee n assessed primarily on Chromosome III and Chr. XV, using Southern hyb ridization to identify these chromosomes and their fragments. At any o ne time, break frequency in wild type is much lower than the cumulativ e frequency of recombination events that occur during meiosis. However , there is suggestive evidence that each break is short-lived, and it is therefore difficult to estimate the total number of breaks that may occur. In rad50S, chromosome breaks accumulate to much higher levels, which are probably broadly consistent with the estimated number of re combination events in wild type. However, since rad50S is substantiall y defective in completing recombination, it is not known for certain i f it initiates events at wild-type frequencies. A surprising feature o f the data is that a strong banding pattern is observed in the fragmen t distribution from broken chromosomes in both strains, implying that at least much of the breakage occurs at specific sites or within short regions. However, with the exception of the rDNA region on Chr. XII, assessment of the genetic map indicates that recombination can occur a lmost anywhere in the genome, although some regions are much hotter th an others. Possible reasons for this apparent paradox are discussed. I t may in part result from breakage levels too low for adequate detecti on in cold regions but may also imply that recombination events are lo calized more than previously realized. Alternatively, there may be a m ore indirect relationship between break sites and the associated recom bination events.