Structural Biology Of The Human THOC1-THOC2-THOC3 Subcomplex And The Study Of The Regulation Of CDK2 Kinase Activity By Speedy A

This thesis contains two parts.The first part is the electron microscopic study of the human THOC1-THOC2-THOC3 subcomplex.Co-expressed in cultured insect cells,the recombinant protein was isolated by two-step affinity purification（Ni-NTA and Strep-Tactin）and one-step gel filtration（Superose）chromatography.Finally,we get a low-resolution model of the human THOC1-THOC2-THOC3 subcomplex identified by negative stain-ing electron microscope and single-particle analysis.This study lays a solid foundation for solving the high-resolution structure of the THO complex and the TREX complex.This study also helps reveal the mechanism of mRNA processing and export.The second part is about the regulation of the CDK2 kinase activity by Speedy A in the meiosis prophaseⅠ.Based on the crystal structure of the human SUN1-Speedy A-CDK2 complex determined by X-ray crysta-llography,we introduce point mutations into Speedy A carried by the expression vector.Then we use E.coli expression systems to co-express the wild-type and mutant Speedy A-CDK2 complex,and after that we detect the phosphorylation of Fox M1 by western blot.Finally,we identify the mutations of E135 and D136 in Speedy A that ablate its ability to activate CDK2 kinase.This study will be helpful for the construction of Speedy A^E134Q/D135N mutant mouse model in future,and it will be helpful for further research on the specific regulation mechanism and physiological significance of SUN1-Speedy A-CDK2 complex during the telomere adhesion nuclear membrane in the meiosis prophaseⅠ.