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Chromatin assembly at kinetochores is uncoupled from DNA replication
R D Shelby1, K Monier, K F Sullivan
1Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
The Journal of Cell Biology
|November 22, 2000
Summary
Centromere identity relies on epigenetic factors, not just DNA. This study shows CENP-A assembly occurs independently of DNA replication, uncoupling histone synthesis from replication timing.
Area of Science:
- Cell Biology
- Epigenetics
- Molecular Biology
Background:
- Centromere specification in metazoans involves epigenetic factors beyond DNA sequence.
- The precise mechanisms establishing centromeric states remain incompletely understood.
Purpose of the Study:
- To investigate the replication timing of human centromeres and CENP-A assembly.
- To determine if CENP-A synthesis and assembly are coupled to DNA replication.
Main Methods:
- Labeling kinetochores with epitope-tagged CENP-A.
- Immunoprecipitation of CENP-A mononucleosomes from synchronized cells pulsed with thymidine.
- Quantitative Western blot and fluorescence microscopy.
- Transfection experiments in the presence of aphidicolin.
Main Results:
- CENP-A-associated DNA replicates during mid-to-late S phase, asynchronously with other genomic regions.
- CENP-A protein synthesis occurs later, in G2 phase.
- CENP-A can assemble into centromeres even without DNA replication.
Conclusions:
- Histone synthesis and assembly at the kinetochore are uncoupled from DNA replication.
- This uncoupling suggests regulated chromatin assembly/remodeling is crucial for epigenetic centromere propagation.