title: Chromatin assembly at kinetochores is uncoupled from DNA replication.
abstract: The specification of metazoan centromeres does not depend strictly on centromeric DNA sequences, but also requires epigenetic factors. The mechanistic basis for establishing a centromeric "state" on the DNA remains unclear. In this work, we have directly examined replication timing of the prekinetochore domain of human chromosomes. Kinetochores were labeled by expression of epitope-tagged CENP-A, which stably marks prekinetochore domains in human cells. By immunoprecipitating CENP-A mononucleosomes from synchronized cells pulsed with [(3)H]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. Quantitative fluorescence microscopy and transient transfection in the presence of aphidicolin, an inhibitor of DNA replication, show that CENP-A can assemble into centromeres in the absence of DNA replication. Thus, unlike most genomic chromatin, histone synthesis and assembly are uncoupled from DNA replication at the kinetochore. Uncoupling DNA replication from CENP-A synthesis suggests that regulated chromatin assembly or remodeling could play a role in epigenetic centromere propagation.
authors.affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: R D
authors.initials: RD
authors.lastname: Shelby
authors.forename: K
authors.initials: K
authors.lastname: Monier
authors.forename: K F
authors.initials: KF
authors.lastname: Sullivan
mesh_terms: D001324:Autoantigens; D002453:Cell Cycle / Q000502:physiology; D002503:Centromere / Q000378:metabolism; D000076247:Centromere Protein A; D002843:Chromatin / Q000502:physiology*; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication / Q000502:physiology*; D000939:Epitopes / Q000235:genetics; D015870:Gene Expression / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D014162:Transfection
chemical_list: D001324:Autoantigens; C000619700:CENPA protein, human; D000076247:Centromere Protein A; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D000939:Epitopes; D012333:RNA, Messenger