title: Chromatin Constrains the Initiation and Elongation of DNA Replication.
abstract: Eukaryotic chromosomal DNA is faithfully replicated in a complex series of cell-cycle-regulated events that are incompletely understood. Here we report the reconstitution of DNA replication free in solution with purified proteins from the budding yeast Saccharomyces cerevisiae. The system recapitulates regulated bidirectional origin activation; synthesis of leading and lagging strands by the three replicative DNA polymerases Pol α, Pol δ, and Pol ε; and canonical maturation of Okazaki fragments into continuous daughter strands. We uncover a dual regulatory role for chromatin during DNA replication: promoting origin dependence and determining Okazaki fragment length by restricting Pol δ progression. This system thus provides a functional platform for the detailed mechanistic analysis of eukaryotic chromosome replication.
authors.affiliation: Molecular Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA; Weill-Cornell Graduate School of Medical Sciences, New York, NY 10065, USA.
authors.forename: Sujan
authors.initials: S
authors.lastname: Devbhandari
authors.affiliation: Molecular Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA; Weill-Cornell Graduate School of Medical Sciences, New York, NY 10065, USA.
authors.forename: Jieqing
authors.initials: J
authors.lastname: Jiang
authors.affiliation: Molecular Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA.
authors.forename: Charanya
authors.initials: C
authors.lastname: Kumar
authors.affiliation: Molecular Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA.
authors.forename: Iestyn
authors.initials: I
authors.lastname: Whitehouse
authors.affiliation: Molecular Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA. Electronic address: remusd@mskcc.org.
authors.forename: Dirk
authors.initials: D
authors.lastname: Remus
mesh_terms: D002843:Chromatin / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism; D004257:DNA Polymerase II / Q000235:genetics / Q000378:metabolism; D004258:DNA Polymerase III / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000378:metabolism; D004271:DNA, Fungal / Q000096:biosynthesis / Q000235:genetics*; D005838:Genotype; D006801:Humans; D009707:Nucleosomes / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D018809:Proliferating Cell Nuclear Antigen / 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
chemical_list: D002843:Chromatin; D004271:DNA, Fungal; D009707:Nucleosomes; C022262:Okazaki fragments; C546594:POL30 protein, S cerevisiae; D018809:Proliferating Cell Nuclear Antigen; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D004256:DNA Polymerase I; D004257:DNA Polymerase II; D004258:DNA Polymerase III; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II
keywords: DNA polymerase; DNA replication; McM2-7; ORC; Okazaki fragment; chromatin; lagging strand; leading strand; replication origin