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Chromosome Research

, Volume 5, Issue 2, pp 132–141 | Cite as

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

  • Kenji Sugimoto
  • Kenji Kuriyama
  • Akiko Shibata
  • Michio Himeno
Article

Abstract

Human centromere protein C (CENP-C), a chromosomal component of the inner plate of kinetochores, was originally identified as one of the centromere auto-antigens. 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 dimerand 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.

autoantigen centromere cross-linking dimerization kinetochore 

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Copyright information

© Chapman and Hall 1997

Authors and Affiliations

  • Kenji Sugimoto
    • 1
  • Kenji Kuriyama
    • 1
  • Akiko Shibata
    • 1
  • Michio Himeno
    • 1
  1. 1.Laboratory of Applied Molecular Biology, Department of Applied BiochemistryUniversity of Osaka PrefectureSakai, OsakaJapan

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