Characterization of internal DNA-binding and C-terminal dimerization domains of human centromere/kinetochore autoantigen CENP-C in vitro: role of DNA-binding and self-associating activities in kinetochore organization.

摘要: Human centromere protein C (CENP-C), a chromosomal component of the inner plate kinetochores, was originally identified as one auto-antigens. In previous study, we showed that it possesses DNA-binding activity in vitro. Recently, centromere-binding suggested at C-terminal region vivo. However, little is known about role CENP-C kinetochore organization. Here, to characterize its biochemical properties, three separate antigenic regions human were expressed Escherichia coli, affinity purified and used South-western blotting chemical cross-linking analyses. We found internal domain composed two kinds elements: ‘core’ flanking ‘stabilizing’ elements support activity. When cross-linked with disuccinimidyl suberate (DSS), N-terminal produced ladder bands dimerand tetramer: exclusively dimer band, whereas not affected all. Dimer formation C-terminus native state also indicated by gel filtration presence conformation-specific autoantibodies patient's sera. These results suggest consists functional units required for ‘kinetochore assembly’: putative oligomerization domain, an dimerization domain.