Centromere protein B assembles human centromeric alpha-satellite DNA at the 17-bp sequence, CENP-B box.

Abstract

We purified 15,000-fold from HeLa cell nuclear extract the centromere antigen that reacts specifically with the 17-bp sequence, designated previously as CENP-B box, in human centromeric alpha-satellite (alphoid) DNA by a two-step procedure including an oligonucleotide affinity column. The purified protein was identified as the centromere protein B (CENP-B) by its mobility on SDS-PAGE (80 kD), and reactivities to a monoclonal antibody raised to CENP-B (bacterial fusion protein) and to anticentromere sera from patients with autoimmune diseases. Direct binding by CENP-B of the CENP-B box sequence in the alphoid DNA has been proved using the purified CENP-B by DNA mobility-shift assay, Southwestern blotting, and DNase I protection analysis. The binding constant of the antigen to the CENP-B box sequence is 6 x 10(8) M-1. DNA mobility-shift assays indicated that the major complex formed between the CENP-B and the DNA contains two DNA molecules, suggesting the importance of the CENP-B/CENP-B box interaction in organization of higher ordered chromatin structures in the centromere and/or kinetochore. Location of DNA binding and dimerization domains in CENP-B was discussed based on the DNA mobility-shift assays performed with a protein fraction containing intact and partial cleavage products of CENP-B.

• Authors: Y Muro, H Masumoto, K Yoda, N Nozaki, M Ohashi, T Okazaki
• Journal: The Journal of cell biology (J Cell Biol) Vol. 116 Issue 3 Pg. 585-96 (Feb 1992) ISSN: 0021-9525 [Print] United States
• PMID: 1730770 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Autoantigens
• CENPB protein, human
• Centromere Protein B
• Chromosomal Proteins, Non-Histone
• DNA, Satellite
• DNA-Binding Proteins

Topics

• Autoantigens
• Base Sequence
• Centromere (metabolism)
• Centromere Protein B
• Chromosomal Proteins, Non-Histone (isolation & purification, metabolism)
• DNA, Satellite (chemistry, metabolism)
• DNA-Binding Proteins
• HeLa Cells
• Humans
• Molecular Sequence Data