CENP-B interacts with CENP-C domains containing Mif2 regions responsible for centromere localization

Recently, human artificial chromosomes featuring functional centromeres have been generated efficiently from naked synthetic alphoid DNA containing CENP-B boxes as a de novo mechanism in a human cultured cell line, but not from the synthetic alphoid DNA only containing mutations within CENP-B boxes, indicating that CENP-B has some functions in assembling centromere/kinetochore components on alphoid DNA. To investigate whether any interactions exist between CENP-B and the other centromere proteins, we screened a cDNA library by yeast two-hybrid analysis. An interaction between CENP-B and CENP-C was detected, and the CENP-C domains required were determined to overlap with three Mif2 homologous regions, which were also revealed to be involved in the CENP-C assembly of centromeres by expression of truncated polypeptides in cultured cells. Overproduction of truncated CENP-B containing no CENP-C interaction domains caused abnormal duplication of CENP-C domains at G(2) and cell cycle delay at metaphase. These results suggest that the interaction between CENP-B and CENP-C may be involved in the correct assembly of CENP-C on alphoid DNA. In other words, a possible molecular linkage may exist between one of the kinetochore components and human centromere DNA through CENP-B/CENP-B box interaction.