title: Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates.
abstract: The 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.
authors.affiliation: Clare Hall Laboratory, Francis Crick Institute, South Mimms, Hertfordshire EN6 3LD, UK.
authors.forename: Christoph F
authors.initials: CF
authors.lastname: Kurat
authors.affiliation: Clare Hall Laboratory, Francis Crick Institute, South Mimms, Hertfordshire EN6 3LD, UK.
authors.forename: Joseph T P
authors.initials: JTP
authors.lastname: Yeeles
authors.affiliation: Lincoln's Inn Fields Laboratory, Francis Crick Institute, London NW1 1AT, UK.
authors.forename: Harshil
authors.initials: H
authors.lastname: Patel
authors.affiliation: Clare Hall Laboratory, Francis Crick Institute, South Mimms, Hertfordshire EN6 3LD, UK.
authors.forename: Anne
authors.initials: A
authors.lastname: Early
authors.affiliation: Clare Hall Laboratory, Francis Crick Institute, South Mimms, Hertfordshire EN6 3LD, UK. Electronic address: john.diffley@crick.ac.uk.
authors.forename: John F X
authors.initials: JFX
authors.lastname: Diffley
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D002843:Chromatin / Q000235:genetics* / Q000378:metabolism; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D004271:DNA, Fungal / Q000096:biosynthesis / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D024001:HMGN Proteins / Q000235:genetics / Q000378:metabolism; D006609:High Mobility Group Proteins / Q000235:genetics / Q000378:metabolism; D051548:Histone Acetyltransferases / Q000235:genetics / Q000378:metabolism; D064110:Minichromosome Maintenance Proteins / Q000235:genetics / Q000378:metabolism; D009707:Nucleosomes / Q000235:genetics* / Q000378:metabolism; D018741:Replication Origin; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D035602:Transcriptional Elongation Factors / Q000235:genetics / Q000378:metabolism; D000099224:p300-CBP-Associated Factor
chemical_list: D000251:Adenosine Triphosphatases; D002843:Chromatin; D004256:DNA Polymerase I; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D024001:HMGN Proteins; D006609:High Mobility Group Proteins; D051548:Histone Acetyltransferases; D064110:Minichromosome Maintenance Proteins; D009707:Nucleosomes; D029701:Saccharomyces cerevisiae Proteins; D035602:Transcriptional Elongation Factors; C541535:FACT protein, S cerevisiae; C483404:INO80 complex, S cerevisiae; C086471:NHP6A protein, S cerevisiae; C086472:NHP6B protein, S cerevisiae; C496551:Esa1 protein, S cerevisiae; C501392:GCN5 protein, S cerevisiae; D000099224:p300-CBP-Associated Factor; C470546:ISW1 protein, S cerevisiae
keywords: DNA replication; biochemistry; chromatin