Modulating Crossover Frequency and Interference for Obligate Crossovers inSaccharomyces cerevisiaeMeiosis

Abstract

Meiotic crossover frequencies show wide variation among organisms. But most organisms maintain at least one crossover per homolog pair (obligate crossover). In Saccharomyces cerevisiae, previous studies have shown crossover frequencies are reduced in the mismatch repair related mutant mlh3D and enhanced in a meiotic checkpoint mutant pch2D by up to twofold at specific chromosomal loci, but both mutants maintain high spore viability. We analyzed meiotic recombination events genome-wide in mlh3D, pch2D, and mlh3D pch2D mutants to test the effect of variation in crossover frequency on obligate crossovers. mlh3D showed $30% genome-wide reduction in crossovers (64 crossovers per meiosis) and loss of the obligate crossover, but nonexchange chromosomes were efficiently segregated. pch2D showed $50% genome-wide increase in crossover frequency (137 crossovers per meiosis), elevated noncrossovers as well as loss of chromosome size dependent double-strand break formation. Meiotic defects associated with pch2Δ did not cause significant increase in nonexchange chromosome frequency. Crossovers were restored to wild-type frequency in the double mutant mlh3D pch2D (100 crossovers per meiosis), but obligate crossovers were compromised. Genetic interference was reduced in mlh3D, pch2D, and mlh3D pch2D. Triple mutant analysis of mlh3D pch2D with other resolvase mutants showed that most of the crossovers in mlh3D pch2D are made through the Mus81-Mms4 pathway. These results are consistent with a requirement for increased crossover frequencies in the absence of genetic interference for obligate crossovers. In conclusion, these data suggest crossover frequencies and the strength of genetic interference in an organism are mutually optimized to ensure obligate crossovers. Meiosis is a reductional division that produces haploid gametes or spores from diploid progenitor cells. Ploidy reduction is achieved by one round of DNA replication, followed by two consecutive nuclear divisions (Meiosis I and II), producing four daughter cells (Roeder 1997). Crossovers promote the formation of chiasma which serves as a physical linkage between two homologs and opposes the spindle generated forces that pull apart the homolog pairs. This opposing set of forces provides the tension necessary to promote proper disjunction of homolog pairs at Meiosis I (Petronczki et al. 2003). Failure to maintain at least one crossover per homolog pair increases the probability of nondisjunction, resulting in aneuploid gametes (Serrentino and Borde 2012). Although crossovers are important for chromosome segregation, nonexchange chromosomes have been observed to segregate accurately forming viable gametes (Hawley et al. 1992;Davis and Smith 2003;Kemp et al. 2004;Newnham et al. 2010;Krishnaprasad et al. 2015). Meiotic crossovers (and noncrossovers) are initiated in S. cerevisiae by 150-170 programmed double-strand breaks (DSBs) formed by an evolutionarily conserved topoisomerase-like protein Spo11 and other

Noncanonical contributions of MutLγ to VDE-initiated crossovers duringSaccharomyces cerevisiaemeiosis

“…However, in pch2∆ mlh3∆ double mutants, COs were reduced 20-35% relative to pch2∆ MLH3 ( Figure 3B; average pch2∆ mlh3∆/pch2∆ = 74 ± 7%), as was previously observed for inserts at URA3 and HIS4 (MEDHI et al 2016). A quantitatively similar MutLg-dependence has also been observed for Spo11-initiated COs in pch2∆ mutants, both genome-wide (pch2∆ mlh3∆ / pch2∆ = 73% (CHAKRABORTY et al 2017) and for individual genetic intervals (pch2∆ / pch2∆ mlh3∆ = 75%, calculated from combined data of NISHANT et al 2008;ZANDERS AND ALANI 2009;AL-SWEEL et al 2017;CHAKRABORTY et al 2017). Thus, the absence of Pch2 increases the MutLg-dependence of VDEinitiated COs at many loci, while decreasing the MutLg-dependence of VDE-initiated COs at HIS4 and of Spo11-initiated COs.…”

“…In contrast to what was seen for VRS inserts at HIS4, where COs were reduced dramatically in mlh3∆ mutants (to ~40% of wild-type levels), COs in the same sequences inserted at all other loci were only modestly affected, with COs in mlh3∆ ranging from ~80% to ~115% of wild type (mean = 91 ± 12%; Figure 2D); NCOs were similarly unaffected ( Figure 2E, F). These results indicate that, in contrast to Spo11-initiated COs, which are reduced about 2-fold in mlh3∆ mutants (WANG et al 1999;KHAZANEHDARI AND BORTS 2000;KIRKPATRICK et al 2000;TSUBOUCHI AND OGAWA 2000;HOFFMANN et al 2003;ARGUESO et al 2004;NISHANT et al 2008;AL-SWEEL et al 2017;CHAKRABORTY et al 2017), most COs at the VDE break sites are formed independent of MutLg, irrespective of the chromosome size, distance from centromere or telomere, or Hop1-enrichment in their vicinity. Thus, at most insert loci in otherwise wild-type cells, VDE-initiated recombination differs from Spo11-initiated recombination and more closely resembles mitotic recombination, in that NCOs are in excess over COs (ESPOSITO 1978;LICHTEN AND HABER 1989;IRA et al 2003;DAYANI et al 2011) and, with the exception of those formed in inserts at HIS4, VDE-initiated COs are largely MutLg-independent.…”

Genome wide analysis of meiotic recombination in yeast: For a few SNPs more

“…Similarly genome wide recombination mapping in mlh2 Δ mutants showed a role for Mlh1‐Mlh2 in regulating the extent of gene conversion tracts 54. Genome wide recombination mapping in msh4 hypomorphs that make fewer crossovers but have normal viability, supported a role for crossover distribution mechanisms in ensuring the obligate crossover 55. Genome wide recombination mapping in pch2 mutants revealed increased crossovers and non‐crossovers as well as loss of chromosome size dependent DSB formation 31.…”

Persistent DNA-break potential near telomeres increases initiation of meiotic recombination on short chromosomes

“…Another unexpected finding is that Pch2 suppresses DSBs around centromeres in late prophase. However, as COs are not enhanced around the centromeres in pch2∆ mutants 43 , Zip1 activity must be sufficient to prevent any deleterious inter-homologue COs in this situation. These findings indicate that several mechanistic layers restrict DSBs and COs at the centromeres, highlighting the importance of limiting COs in this region.…”

“…One possible function of long-lived hotspots is to increase the window of opportunity for DSB formation on short chromosomes. Short chromosomes exhibit elevated recombination density in many organisms 25,[42][43][44][45][46] . In yeast, this bias is already apparent at the level of DSB formation [47][48][49][50] and is likely driven by two independent mechanisms, both of which remain poorly understood.…”