Abstract
SummaryThe integrity of eukaryotic genomes requires rapid and regulated chromatin replication. How this is accomplished is still poorly understood. Using purified yeast replication proteins and fully chromatinized templates, we have reconstituted this process in vitro. We show that chromatin enforces DNA replication origin specificity by preventing non-specific MCM helicase loading. Helicase activation occurs efficiently in the context of chromatin, but subsequent replisome progression requires the histone chaperone FACT (facilitates chromatin transcription). The FACT-associated Nhp6 protein, the nucleosome remodelers INO80 or ISW1A, and the lysine acetyltransferases Gcn5 and Esa1 each contribute separately to maximum DNA synthesis rates. Chromatin promotes the regular priming of lagging-strand DNA synthesis by facilitating DNA polymerase α function at replication forks. Finally, nucleosomes disrupted during replication are efficiently re-assembled into regular arrays on nascent DNA. Our work defines the minimum requirements for chromatin replication in vitro and shows how multiple chromatin factors might modulate replication fork rates in vivo.
Highlights
Eukaryotic genomes are packaged into nucleosomes comprising 147 bp of duplex DNA wrapped around a histone octamer containing two copies each of the four core histones (H2A, H2B, H3, and H4) (Luger et al, 1997)
Analysis of the nucleosome arrays produced by micrococcal nuclease (MNase) digestion showed a high density of evenly spaced nucleosomes in the population (Figure S1B)
We assembled chromatin on a 2.8-kb fragment of yeast DNA from the TRP1-GAL3 locus with the ARS1 replication origin at its center, which was attached at one end to magnetic beads via a biotin-streptavidin linkage (Figure 1A; Figures S1A and S1B)
Summary
Eukaryotic genomes are packaged into nucleosomes comprising 147 bp of duplex DNA wrapped around a histone octamer containing two copies each of the four core histones (H2A, H2B, H3, and H4) (Luger et al, 1997). Histones are highly basic proteins and nucleosomes are very stable structures, requiring, for example, high salt concentrations for their removal from chromatin Within this context, the replication machinery must define sites of replication initiation (origins), load the MCM replicative helicase, and activate it by converting it to the CMG (Cdc45MCM-GINS) complex. Many ‘‘chromatin factors’’ have been described that affect chromatin structure or dynamics including histone chaperones, nucleosome remodelers, and enzymes that covalently modify histone subunits (Campos and Reinberg, 2009; Swygert and Peterson, 2014). The roles of these proteins in transcription, DNA repair, and DNA damage signaling have been well studied. Roles for these factors in chromatin replication are still poorly defined
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