Centromeres coincide with the first set of heterochromatic replication origins. K. Ahmad ², J. Platero ^1,2, S. Henikoff ^1,2. 1) Howard Hughes Medical Institute; 2) Fred Hutchinson Cancer Res Ctr, Seattle, WA.

   Specialized nucleosomes containing distinct H3-like histones are found at the centromeres of eucaryotic chromosomes. Centromeres are often embedded in heterochromatin, and there have been suggestions that heterochromatin has some mitotic functions. We wondered if heterochromatin facilitates the deposition of specialized nucleosomes at the centromere. To this end, we characterized in detail the timing of replication at and around centromeres. We used antibodies raised against the Drosophila centromeric H3-like histone Cid (Centromere identifier) protein to mark centromeres throughout interphase in Kc167 line cells, and labelled newly replicated DNA with either BrdU or pulses of histone-GFP protein. All four centromeric spots (each representing a set of homologous chromosomes) appear to replicate together in mid-S-phase, just as the euchromatic portion of the genome is completing replication. Very few, if any, non-centromeric heterochromatic regions have replicated by this time. This specific window of replication may coincide with the cell-cycle limited expression of the cid promoter. To directly address whether replication in this window is required for deposition of Cid at centromeres, we are producing CidGFP fusion protein from a heat-shock inducible promoter and following its localization to centromeres. These experiments will address the role of replication timing and specialized centromeric nucleosomes in maintaining the centromere.