Functional Research Of Scc4 In Meiosis Of Saccharomyces Cerevisiae

Meiosis is the essential process of gametogenesis,which is required for sexual reproduction.Aberrant meiosis is the leading cause of human infertility,abortion,stillbirth and birth defects.However,the molecular mechanism is still not well understood.Comprehensive understanding the mechanism of meiosis is of great significance for preventing birth defects and improving human reproductive health.Sister chromatid cohesion is a key event during mitosis and meiosis,and it is essential to ensure the correct segregation of chromosomes.Sister chromatid cohesion is mainly mediated by the cohesin complex,which is recruited and loaded onto chromosomes by cohesin loader complex at G1/S phase.In most organisms,including human and budding yeast Saccharomyces.cerevisiae,cohesin loader complex is composed of Scc2 and Scc4.Recent studies showed that cohesin loader complex also plays critical roles in multiple biological processes such as the regulation of gene expression.Mutation of cohesin loader complex results in Cornelia de Lange syndrome in humans,and Mau2(Scc4)deletion in mouse leads to craniofacial loss.Several studies have also indicated that the two subunits may have different functions.For example,in Saccharomyces cerevisiae scc4 mutants have decreased cohesin level around pericentromeres,however,scc2 mutants have decreased cohesin level on chromosome arms during mitosis.In addition,Scc2 is required for the expression of REC8,which encodes a meiosis-specific cohesin subunit.In order to investigate the roles of Scc4 during meiosis,we constructed Saccharomyces cerevisiae scc4 mutants,including pCLB2-SCC4 in which Scc4 is specifically depleted during meiosis,scc4-m35 and pCLB2-SCC4/scc4-m35 with decreased the loading efficiency of cohesin.Studies showed that meiosis is severely affected in pCLB2-SCC4,including significantly decreased sporulation level and spore viability,and the meiosis nuclear division is also delayed.Rad51 can binds to the singlestrand DNA processed from DNA double-strand breaks(DSBs).Thus,we applied cell spread and immune-fluorescence to analyze the number and dynamics of Rad51 foci,and found that the repair of meiotic DSBs is remarkably delayed in pCLB2-SCC4 mutants.One dimension DNA gel electrophoresis and Southern blot analysis also revealed that the level of meiotic DSBs in pCLB2-SCC4 mutants at the meiotic recombination hotspot HIS4-LEU2 was reduced to 40%of the wild type level.Meiotic DSBs can be repaired as crossover or non-crossover.Further studies at HIS4-LEU2 locus with DNA gel electrophoresis and Southern blot analysis showed that pCLB2-SCC4 specifically decreased the level of crossover,but the level of non-crossover was maintained at wild type level.Consistent with the opinion that crossover are regulated by homologous chromosome synapsis and chromosome axes,immuno-fluorescence staining results showed that both homologous synapsis and chromosome axes are impaired in pCLB2-SCC4.The two mutants with decreased cohesion loading efficiency also showed similar meiotic defects although less severe the phenotypes.These results indicated that Scc4 plays critical roles in meiosis.And based on these results,we proposed that Scc4 may affect DNA double-strand breaks and recombination repair by regulating the formation of meiotic chromosome axis and also homologous synapsis.Meiotic recombination defects lead to decreased sporulation level and spore viability.This is very useful for our understanding of the molecular mechanisms of meiosis.