Preliminary Study On Meiotic Recombination Regulated By The Width Of Synaptonemal Complex

A critical role of meiosis in eukaryote sexual reproduction is to generate gametes with half chromosome number.It is accomplished by a single round of DNA replication and two rounds of successive chromosome separation.During meiosis homologous chromosomes recognize each other and pair to form synaptonemal complex in wide of species.The structure of the synaptonemal complex between different species is highly conserved,which consists of two parallel lateral elements and central region.The central region is composed of transverse filaments and central elements.Studies have found that the width of synaptonemal complex is about 100 nm among different species.The width of synaptonemal complex can be changed by increasing or decreasing the length of transverse filaments.For example,Zip1 is the transverse filaments of Saccharomyces cerevisiae.By deleting or inserting the Coiledcoil structure in the middle of the Zipl protein,the width of the synaptonemal complex can be reduced or increased.When the width of the synaptonemal complex is reduced,the crossover frequency of Saccharomyces cerevisiae decreases.How the width of synaptonemal complex regulates meiotic recombination remains unclear.In this study,we constructed some mutants in Saccharomyces cerevisiae and analyzed the molecular mechanism of meiosis recombination regulated by synaptonemal complex width in detail by genetic and cytological methods.Firstly,we constructed four mutants,including zip1-M588 and zip1-M1173 mutants,whose synaptonemal complex width were reduced by 20.18 nm and 29.40 nm respectively,and zip1-2H2 and zip1-3H2 mutants,whose synaptonemal complex width were increased by 33.46 nm and 56.60 nm respectively.Preliminary studies have found that whether the width of synaptonemal complex decreases or increases,it will seriously affect the meiosis of Saccharomyces cerevisiae.Compared with the wild type,the meiosis process of the zip1-M588,zip1-M1173,zip1-2H2 and zip1-3H2 were delayed,and the sporulation efficiency was reduced to 91.09%,80.57%,87.93%and 88.18%respectively,and the spore viability was reduced to 80.04%,51.10%,78.12%and 48.94%respectively.Furthermore,we explored the molecular mechanism of meiosis recombination regulated by synaptonemal complex width.Meiosis recombination starts with programmed DNA double-strand breaks(DSB).Recombinase Rad51,as a classical maker of DSB,participates in the homologous template strands search and invasion process during the repair of DSB.Through chromosome spreading and immunefluorescence imaging technology,we analyzed the dynamic changes of Rad51 foci in different mutants during meiosis.The results showed that changing the width of synaptonemal complex does not affect the level of DSB,but it will significantly affect the repair of DSB.Crossover is critical for meiosis.E3 ligase Zip3 can be used as a marker for Crossover in Saccharomyces cerevisiae.Immuno-fluorescence showed that the number of Zip3 foci in the zip1-M1173 and zip1-2H2 mutants were reduced to 30.50 and 43.79(59.95 in the wild type),indicating that the width of the synaptonemal complex is necessary for meiotic crossover.However,the number of Zip3 foci in the zip1-M588 mutant is similar to that of the wild type,and the specific molecular mechanism needs to be further explored.Since we changed the width of synaptonemal complex by truncating or increasing the coiled coil domain of Zip1,we further analyzed whether the recombination defects was caused by the changes of synaptonemal complex width or changes of Zip1 Coiledcoil domain.Firstly,we constructed zip1-M1173::GFP mutant by replacing the coiledcoil domain of Zip1 with GFP,who has no coiled-coil domain.Meanwhile,the coiled coil domain of Zipl was replaced by the coiled-coil domain of mouse tropomyosin Tpm1 to obtain zip1-M1173::Tpm1 mutant.We further found that the width of synaptonemal complex of zip1-M1173::GFP and zip1-M1173::Tpm1 mutants was similar to that of wild type,and there were obvious defects during meiosis in both mutants.For example,the meiosis process of zip1-M1173::GFP and zip1M1173::Tpm1 mutants were delayed for about 3 hours compared with the wild type,and the spore viability was reduced to 50.69%and 10.76%respectively.Interestingly,the Zip3 foci of zip1-M1173::Tpm1(39.4)is higher than that of zip1-M1173 mutant,but lower than wild type.These results suggest that the maintenance of synaptonemal complex width is critical for meiosis recombination,but the coiled-coil domain of Zip1 also plays an important role.In this study,we explored the effect of synaptonemal complex width on meiotic reorganization,which provides a new perspective and evidence for further revealing the molecular mechanism of meiosis.