SURPRISING DEFICIENCY OF CENP-B BINDING-SITES IN AFRICAN-GREEN MONKEYALPHA-SATELLITE DNA - IMPLICATIONS FOR CENP-B FUNCTION AT CENTROMERES

Centromeres of mammalian chromosomes are rich in repetitive DNAs that are packaged into specialized nucleoprotein structures called heteroch romatin. In humans, the major centromeric repetitive DNA, alpha-satell ite DNA, has been extensively sequenced and shown to contain binding s ites for CENP-B, an 80-kDa centromeric autoantigen. The present report reveals that African green monkey (AGM) cells, which contain extensiv e a-satellite arrays at centromeres, appear to lack the well-character ized CENP-B binding site (the CENP-B box). We show that AGM cells expr ess a functional CENP-B homolog that binds to the CENP-B box and is re cognized by several independent anti-CENP-B antibodies. However, three independent assays fail to reveal CENP-B binding sites in AGM DNA, Me thods used include a gel mobility shift competition assay using purifi ed AGM alpha-satellite, a novel kinetic electrophoretic mobility shift assay competition protocol using bulk genomic DNA, and bulk sequencin g of 76 AGM alpha-satellite monomers. Immunofluorescence studies revea l the presence of significant levels of CENP-B antigen dispersed diffu sely throughout the nuclei of interphase cells. These experiments reve al a paradox, CENP-B is highly conserved among mammals, yet its DNA bi nding site is conserved in human and mouse genomes but not in the AGM genome. One interpretation of these findings is that the role of CENP- B may be in the maintenance and/or organization of centromeric satelli te DNA arrays rather than a more direct involvement in centromere stru cture.