Centromere protein B assembles human centromeric alpha-satellite DNA at the 17-bp sequence, CENP-B box.
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.
Muro
Y
Y
Department of Dermatology, Nagoya University School of Medicine, Japan.
Masumoto
H
H
Yoda
K
K
Nozaki
N
N
Ohashi
M
M
Okazaki
T
T
D001324:Autoantigens; D001483:Base Sequence; D002503:Centromere / Q000378:metabolism*; D051936:Centromere Protein B; D002868:Chromosomal Proteins, Non-Histone / Q000302:isolation & purification / Q000378:metabolism*; D004276:DNA, Satellite / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data
D001324:Autoantigens; C497926:CENPB protein, human; D051936:Centromere Protein B; D002868:Chromosomal Proteins, Non-Histone; D004276:DNA, Satellite; D004268:DNA-Binding Proteins