Chromatin assembly at kinetochores is uncoupled from DNA replication

The specification of metazoan centromeres does not depend strictly on centr omeric DNA sequences, but also requires epigenetic factors. The mechanistic basis for establishing a centromeric "state" on the DNA remains unclear. I n this work, we have directly examined replication timing of the prekinetoc hore domain of human chromosomes. Kinetochores were labeled by expression o f epitope-tagged CENP-A, which stably marks prekinetochore domains in human cells. By immunoprecipitating CENP-A mononucleosomes from synchronized cel ls pulsed with [H-3]thymidine we demonstrate that CENP-A-associated DNA is replicated in mid-to-late S phase. Cytological analysis of DNA replication further demonstrated that centromeres replicate asynchronously in parallel with numerous other genomic regions. In contrast, quantitative Western blot analysis demonstrates that CENP-A protein synthesis occurs later, in G2. Q uantitative fluorescence microscopy and transient transfection in the prese nce of aphidicolin, an inhibitor of DNA replication, show that CENP-A can a ssemble into centromeres in the absence of DNA replication. Taus, unlike mo st genomic chromatin, histone synthesis and assembly are uncoupled from DNA replication at the kinetochore. Uncoupling DNA replication from CENP-A syn thesis suggests that regulated chromatin assembly or remodeling could play a role in epigenetic centromere propagation.