In-vitro Reconstitution And Interaction Analysis Of Kinetochore CENP-C/H/K Complex

In the process of mitosis,the accurate separation of chromosomes requires coordinated operations of many subcellular structures.The kinetochore,which located on both sides of centromere,is an important participant of this process.The deletion or abnormality of kinetochore proteins will lead to the failure of spindle microtubules binding to chromosomes,the delay of polar-ward chromosome movement,and other catastrophic consequences such as chromosome mis-segregation and aneuploidy.Human kinetochore complex contains two parts:the inner kinetochore and the outer kinetochore.The inner kinetochore binds to CENP-A,a centromeric histone H3 variant,and links to the centromere DNA.The inner kinetochore distributed into five functional groups as follows:CENP-C,CENP-H/I/K/M,CENP-L/N,CENP-T/W/S/X and CENP-O/P/Q/U/R.The outer kinetochore consists of three subcomplexes:KNL1,MIS12and NDC80.The outer kinetochore played an important part in microtubule attachment and mitotic checkpoint regulation.Although great progress has been made,the structure and assembly mechanism of kinetochore complex is still not well understood.In this thesis,we focused on the interaction analysis and structure characters of CENP-C,CENP-H and CENP-K,which are important subunits of the inner kinetochore.We expressed and purified CENP-C and CENP-K proteins of different lengths by molecular cloning and recombinant expression,and reconstituted CENP-H/K and CENP-C/H/K complexes in vitro.Through the in vitro reconstitution,we found that the CENP-H/K subcomplex formed a tight heterodimer through the interaction between their C-terminal fragments.By Pull-Down assay,we found that CENP-C^277-325 is the key motif for CENP-H/K binding while CENP-H mediates the interaction between CENP-H/K subcomplex and CENP-C.In addition,the ability of CENP-H^107-C/K^85-C subcomplex binding to CENP-C was similar to full-length CENP-H/K subcomplex,indicating that the interaction between CENP-C and CENP-H/K complex mainly depends on the C-terminal fragments of CENP-H/K complex.Through the interaction analysis and recombination optimization,we gained proteins and complexes in good state.We tried crystallization screening of CENP-C,CENP-K,CENP-H/K and CENP-C/H/K complexes.At present,we found some cluster crystals of CENP-C^208-400/H^FL/K^FL complex.The process of crystallization optimization is still on going.This thesis partly reveals the interactions mechanism among the kinetochore protein CENP-C,CENP-H and CENP-K,and provided an important basis for further structural and functional studies.