Nucleosomes influence multiple steps during replication initiation
- Howard Hughes Medical Institute, Massachusetts Institute of Technology, United States
- University of Massachusetts Medical School, United States
- Duke University Medical Center, United States
Abstract
Eukaryotic replication origin licensing, activation and timing are influenced by chromatin but a mechanistic understanding is lacking. Using reconstituted nucleosomal DNA replication assays, we assessed the impact of nucleosomes on replication initiation. To generate distinct nucleosomal landscapes, different chromatin-remodeling enzymes (CREs) were used to remodel nucleosomes on origin-DNA templates. Nucleosomal organization influenced two steps of replication initiation: origin licensing and helicase activation. Origin licensing assays showed that local nucleosome positioning enhanced origin specificity and modulated helicase loading by influencing ORC DNA binding. Interestingly, SWI/SNF- and RSC-remodeled nucleosomes were permissive for origin licensing but showed reduced helicase activation. Specific CREs rescued replication of these templates if added prior to helicase activation, indicating a permissive chromatin state must be established during origin licensing to allow efficient origin activation. Our studies show nucleosomes directly modulate origin licensing and activation through distinct mechanisms and provide insights into the regulation of replication initiation by chromatin.
Article and author information
Author details
Funding
Howard Hughes Medical Institute (Investigator)
- Stephen P Bell
National Institute of General Medical Sciences (GM54096 and GM104097)
- David M MacAlpine
- Craig L Peterson
American Cancer Society (123700-PF-13-071-01-DM)
- Ishara F Azmi
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2017, Azmi et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Nucleosomes influence multiple steps during replication initiation
eLife 6:e22512.
https://doi.org/10.7554/eLife.22512
