In-vitro Reconstitution Reveals The Novel Mechanism Of How Nucleosomes And Histones Recuit Chromosome Protect Protein Sgo1

The accurate separation of chromosomes is essential for the faithful distribution of genetics to daughter cell in cell division.During mitosis,the mis-segregation of chromosomes will lead to the aneuploidy and chromosome instability,and then cause cell malignant transformation or death.Sgo1(shugoshin 1)protein has been proved to play an important role in chromosome segregation and is called the"protector"for centromeric Cohesin complex.Sgo1 locates in the centromere and maintains the inner-force in the centromeric region together with Cohesin complex,which makes the metaphase chromosomes exhibit a typical"X"shape,thus ensures the stability of eukaryotic chromosomes in mitosis and meiosis.Mitotic kinase Bub1 phosphorylates threonine(p T120)at the centromeric histone H2A,while phosphorylated histone H2Ap T120 recruits Sgo1 into the centromere.However,the molecular mechanism and structural basis of Sgo1recruitment are still unclear.In this thesis,we try to analyze the recruitment mechanism of how nucleosome and histone recruit Sgo1 complex by in vitro reconstitution and biochemical methods.In this study,we successfully reconstructed the Xenopus nucleosome in vitro by salt gradient dialysis,and identified the nucleosome assembled by negative staining and Cryo-EM.To mimic the phosphorylation of histone H2A in vitro,we introduced T120D mutation on histone H2A into the assembled nucleosome,and verified the interaction between histone H2A and Sgo1 by electrophoretic mobility shift assay(EMSA).And interestingly,nucleosome composition DNA did not participate in the interaction between Sgo1 and nucleosome.We then reconstituted the heterodimer of H2A-H2B and H2AT120D-H2B in vitro,and confirmed the direct interaction between histone H2A/B and Sgo1 by in vitro binding assay(Pull down).Through the recombinant protein expression and in-vitro reconstitution system,we obtained H2A-H2B-sgo1^554-663 and H2A^T120D-H2B-sgo1^554-663protein complexes and screened the crystals.The crystal structure would be helpful to further understand how Sgo1 is recruited into chromosome.In this study,the interaction between Sgo1 and nucleosome was successfully identified in vitro,the interaction region of Sgo was further determined,and the protein complex was obtained with good properties.Secondly,our results revealed a novel mechanism for histone recruitment of Sgo1 that both histone H2A-H2B and H2A^T120D-H2B heterodimer recruit Sgo1 independently in-vitro,which questions the previous conclusion that phosphorylated H2A in a complete nucleosome are necessary to recruit Sgo1.Our result suggests a more specific recruitment mechanism and re-evaluates the importance of threonine at position 120 of the histone H2A.Our studies provide a new insight for understanding how Sgo1 is recruited into centromere.