Purification and characterization of a CENP-B homologue protein that binds to the centromeric K-type repeat DNA of Schizosaccharomyces pombe

Purification and characterization of a CENP-B homologue protein that binds to the centromeric K-type repeat DNA of Schizosaccharomyces pombe

^*Memorial Sloan–Kettering Cancer Center, New York, NY 10021; and ^†Department of Molecular Biology, Roswell Park Cancer Institute, Buffalo, NY 14263

Abstract

We have purified and characterized a novel 60-kDa protein that binds to centromeric K-type repeat DNA from Schizosaccharomyces pombe. This protein was initially purified by its ability to bind to the autonomously replicating sequence 3002 DNA. Cloning of the gene encoding this protein revealed that it possesses significant homology to the mammalian centromere DNA-binding protein CENP-B and S. pombe Abp1, and this gene was designated as cbh ⁺ (CENP-B homologue). Cbh protein specifically interacts in vitro with the K-type repeat DNA, which is essential for centromere function. The Cbh-binding consensus sequence was determined by DNase I footprinting assays as PyPuATATPyPuTA, featuring an inverted repeat of the first four nucleotides. Based on its binding activity to centromeric DNA and homology to centromere proteins, we suggest that this protein may be a functional homologue of the mammalian CENP-B in S. pombe.

Footnotes

↵ ‡ To whom reprint requests should be addressed at: Graduate Program in Molecular Biology, Memorial Sloan–Kettering Cancer Center, 1275 York Avenue, Box 97, New York, NY 10021.
Jerard Hurwitz
Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. U86754).
ABBREVIATIONS:

ARS,

autonomously replicating sequence;

cc2,

central core sequence of centromere 2;

K-d,

K"-repeat d fragment