Functional cloning of centromere protein B (CENP-B) box-enriched alphoid DNA repeats utilizing the sequence-specific DNA binding activity of human CENP-Bin vitro

Abstract

The centromere is a distinctive portion of the chromosome consisting of 'centromere DNA' and 'centromere proteins'. Recently, a direct molecular interaction was discovered between human centromere protein B (CENP-B) and human centromeric alphoid repeats. This enabled us to isolate the CENP-B-targeted centromeric DNA sequences by positively utilizing the biologic activity of CENP-B in vitro. In the previous model experiment, we found that oligonucleotides covering the CENP-B binding sequences were enriched by the DNA immunoprecipitation procedure. Here we apply the same technique to the direct isolation of a functional part of human centromeric DNA from a genomic DNA library. Restriction digestion of two isolated clones showed the typical repeating pattern of an alphoid family that is known to localize at the centromeric region of all human chromosomes. Sequence analysis showed that these two clones frequently contain the authentic CENP-B binding motif, CTTCGTTGGAAACGGGA, or a new one with one base replaced, CTTCGTTGGAAACGGGT. The frequent distribution of these motifs suggests that the isolated sequences are directly involved in the organization of centromeric heterochromatin at the primary constriction in conjunction with CENP-B.