utation of a Meiosis-Specific MutS creases Crossing Over Not Mismatch Correction

acdonald and G. Shirleen Roeder epartment of Biology Yale University New Haven, Connecticut 06520-8103 SH4 is a novel meiosis-specific gene required for wrid-type levels of spore viability in S. cerevisiae. The edicted product of the MSH4 gene is homologous the MutSfamilyof proteins; however, mshrl-null mu- tants have no apparent defect in mismatch repair. msh4 mutant strains display wild-type levels of gene conversion and postmeiotic segregation, but they show a reduction in crossing over and a resultant in- crease in nondisjunction of homologous chromo- mes at meiosis I. lmmunofluorescence experiments emonstrate that the Msh4 protein is localized to dis- crete sites on pachytene chromosomes. We propose that Msh4 interacts with a recombination intermediate to influence its resolution. During meiosis, two successive nuclear divisions generate four haploid progeny from asinglediploid cell. The meiosis Ii division resembles mitotic chromosome segregation, but the meiosis I division is unique in that homologous chro- mosomes disjoin from each other. Correct orientation of chromosomes on the meiosis I spindle is assured by the presence of connections, known as chiasmata, between homologs. Chiasmata are the physical manifestations of crossing over, and their establishment is dependent on a conjunction of events unique to meiotic prophase. These include high levelsof recombination, chromatin condensa- tion, and the formation of a novel proteinaceous entity called the synaptonemal complex (SC). Cytological studies of the SC reveal strong evolutionary conservation (von Wettstein et al., 1984). Following mei- otic DNA synthesis, chromatin in each pair of sister chro- matids becomes organized as a series of loops, attached at their base to a linear proteinaceous structure called an axial element. This reorganization may still be underway as cells enter zygotene, when homologs align and SC for- mation begins. At pachytene, full synapsis occurs, and the axial elements become closely apposed along the entire length of the bivalent, forming the lateral elements of the SC. At diplotene, the SC proteins disassemble and the chromosomes desynapse. 167 a number of organisms that undergo meiotic crossing over, electron-dense structures, called recombination nod- ules, have been observed in association with the SC (Car- penter. 1988). Late in pachytene, the frequency and distribu- tion of these structures correlates well with those of cross- overs, leading to the hypothesis that the structures are sites of multiprotein complexes that mediate exchang@. Recombi- nation nodules identified in zygotene or early pachytene are usually more abundant and a different shape than those present at later stages. It has been suggested that the early nodules mark sites of strand exchange, whereas late nod- ules represent only those strand exchange events that will oe resolved as crossovers (Carpenter, 1988). Meiosis-specific double-strand breaks have been identi- fied at a number of recombinational hotspots in yeast and are thought to initiate some or all meiotic recombination events (Petes et al., 1991). The 5’ termini of such breaks are normally recised to generate single-stranded tails (Sun et al., 1991; Bishop 1992) that may serve as agents of strand invasion. In most models for the molecular mech- anism of recombination, the strand exchange intermediate consists of a region hybrid or heteroduplex DNA in asso- ciation with one or two Holliday junctions (Qrr Szostak, 1985; Sun et al., 1991). Base pair within the heteroduplex are sometimes corrected, re- sulting in gene conversion. If mismatch correction does not occur, postmeiotic segregation (PMS) resuits. Holiiday junctions are resolved to give either a parental or recom- binant configuration of flanking markers. Approximately 50% of all gene conversion events are accompanied by reciprocal exchange, suggesting that gene conversion and crossing over are associated pro- cesses (Petes et al., 1991). Many meiotic mutants of yeast affect both reciprocal and nonreciprocal recombination, roviding additional evidence that the two types of events are related (Petes et al., 1991). Molecular models of re- combination depict resolution as a stoch (Crr-Weaver and Szostak, 1985; Sun et vitro studies of Holliday junction-cleavi cate that local DNA sequence is the of the direction of resolution (West, 1992). However, the phenomenon of crossover interference (Jones, 1984) sug- gests that the resolution of Holliday junctions in viva is affected by other factors. In addition, there are several meiotic mutants in Drosophila and yeast in which recipro- cal recombination is reduced, but gene conve is unaf- fected (Carpenter, 1988; Engebrecht et al., 1 Sym al., 1993; Sym and Roeder, 1994). The phenotypes of these mutants suggest that crossing over involves addi- tional proteins not required for simple gene conversion. We have identifed a novel meiosis-specific gene named SW4 (Burns et al., 1994). The Msh4 protein shows exten- save homology to the bacterial MutS proteins, which initiate mismatch repair by binding directly to the mispaired bases (Modrich, 1991). However, msh4 mutants have no appar- ent defect in mismatch correction. Instead, they display a specific defect in reciprocal recombination, leading to the nondisjunction of homologous chromosomes at meio- sis I and consequent spore inviability. ihe localizes to discrete sites on meiotic chromosomes, rais- ing the possibility that Msh4 is a component of recambina- tion nodules.