Nucleosomes influence multiple steps during replication initiation.
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.
Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, United States.
Ishara F
0000-0001-6280-3149
IF
Azmi
Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, United States.
Shinya
S
Watanabe
Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, United States.
Michael F
MF
Maloney
Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, United States.|Center for Genomic Integrity, Institute for Basic Science, Ulsan, South Korea.
Sukhyun
S
Kang
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, United States.|Program in Computational Biology and Bioinformatics, Duke University, Durham, United States.
Jason A
0000-0003-2945-6282
JA
Belsky
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, United States.
David M
DM
MacAlpine
Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, United States.
Craig L
CL
Peterson
Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, United States.
Stephen P
0000-0002-2876-610X
SP
Bell
D004247:DNA / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism; D004261:DNA Replication; D009707:Nucleosomes / Q000378:metabolism*; D011485:Protein Binding; D018741:Replication Origin; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000502:physiology
D009707:Nucleosomes; D004247:DNA; D004265:DNA Helicases
DNA Replication; Eukaryotic; Helicase; S. cerevisiae; biochemistry; chromatin remodeling enzyme; chromosomes; genes; nucleosome; origin licensing