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.
Human centromere protein C (CENP-C), a chromosomal component of the inner plate of kinetochores, was originally identified as one of the centromere autoantigens. In a previous study, we showed that it possesses DNA-binding activity in vitro. Recently, centromere-binding activity was suggested at the C-terminal region in vivo. However, little is known about the role of CENP-C in kinetochore organization. Here, to characterize its biochemical properties, three separate antigenic regions of human CENP-C were expressed in Escherichia coli, affinity purified and used in South-western blotting and chemical cross-linking analyses. We found that the internal DNA-binding domain was composed of two kinds of elements: the 'core' and two flanking 'stabilizing' elements that support the activity. When cross-linked with disuccinimidyl suberate (DSS), the N-terminal region produced the ladder bands of dimer and tetramer: the C-terminal region exclusively produced the dimer band, whereas the internal region was not affected at all. Dimer formation at the C-terminus in the native state was also indicated by gel filtration and the presence of conformation-specific autoantibodies in the patient's sera. These results suggest that human CENP-C consists of three functional units required for 'kinetochore assembly': a putative N-terminal oligomerization domain, an internal DNA-binding domain and a C-terminal dimerization domain.
Sugimoto
K
K
Department of Applied Biochemistry, University of Osaka Prefecture, Japan.
Kuriyama
K
K
Shibata
A
A
Himeno
M
M
D001324:Autoantigens / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002503:Centromere; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003432:Cross-Linking Reagents; D004247:DNA / Q000378:metabolism*; D019281:Dimerization; D018604:Epitope Mapping; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D018386:Kinetochores / Q000737:chemistry*; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins; D013388:Succinimides
D001324:Autoantigens; D002868:Chromosomal Proteins, Non-Histone; D003432:Cross-Linking Reagents; D010455:Peptides; D011993:Recombinant Fusion Proteins; D013388:Succinimides; C060905:centromere protein C; D004247:DNA; C019358:disuccinimidyl suberate