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.
Highlights
The eukaryotic genome is packaged into a condensed form known as chromatin that presents a barrier to DNA-associated processes
The development of fully-reconstituted replication-initiation assays (Yeeles et al, 2015, 2017) has opened the way to biochemically investigate the interactions between the replication-initiation machinery and nucleosomes
We have used origin-containing nucleosomal DNA templates assembled in the presence of different chromatin-remodeling enzymes (CREs) to investigate how origin-proximal nucleosomes affect replication initiation
Summary
The eukaryotic genome is packaged into a condensed form known as chromatin that presents a barrier to DNA-associated processes. Chromatin is primarily composed of nucleosomes, each of which consists of ~147 base pairs of DNA wrapped around a histone octamer. The location and modification state of nucleosomes is dynamic, regulates access to the DNA and partitions the genome into distinct chromatin states (Clapier and Cairns, 2009). Nucleosome positioning and modifications influence all DNA processes including replication, transcription, repair and recombination. Maintaining appropriate chromatin states across the genome is critical for cellular viability (Hargreaves and Crabtree, 2011). There is a growing wealth of knowledge concerning the impact of nucleosomes on gene expression, significantly less is known about the role of nucleosomes in regulating DNA replication
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