title: How the Eukaryotic Replisome Achieves Rapid and Efficient DNA Replication.
abstract: The eukaryotic replisome is a molecular machine that coordinates the Cdc45-MCM-GINS (CMG) replicative DNA helicase with DNA polymerases α, δ, and ε and other proteins to copy the leading- and lagging-strand templates at rates between 1 and 2 kb min-1. We have now reconstituted this sophisticated machine with purified proteins, beginning with regulated CMG assembly and activation. We show that replisome-associated factors Mrc1 and Csm3/Tof1 are crucial for in vivo rates of replisome progression. Additionally, maximal rates only occur when DNA polymerase ε catalyzes leading-strand synthesis together with its processivity factor PCNA. DNA polymerase δ can support leading-strand synthesis, but at slower rates. DNA polymerase δ is required for lagging-strand synthesis, but surprisingly also plays a role in establishing leading-strand synthesis, before DNA polymerase ε engagement. We propose that switching between these DNA polymerases also contributes to leading-strand synthesis under conditions of replicative stress.
authors.affiliation: The Francis Crick Institute, Clare Hall Laboratory, South Mimms, Potters Bar, Hertfordshire EN6 3LD, UK.
authors.forename: Joseph T P
authors.initials: JTP
authors.lastname: Yeeles
authors.affiliation: The Francis Crick Institute, Clare Hall Laboratory, South Mimms, Potters Bar, Hertfordshire EN6 3LD, UK.
authors.forename: Agnieska
authors.initials: A
authors.lastname: Janska
authors.affiliation: The Francis Crick Institute, Clare Hall Laboratory, South Mimms, Potters Bar, Hertfordshire EN6 3LD, UK.
authors.forename: Anne
authors.initials: A
authors.lastname: Early
authors.affiliation: The Francis Crick Institute, Clare Hall Laboratory, South Mimms, Potters Bar, Hertfordshire EN6 3LD, UK. Electronic address: john.diffley@crick.ac.uk.
authors.forename: John F X
authors.initials: JFX
authors.lastname: Diffley
mesh_terms: D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D004257:DNA Polymerase II / Q000235:genetics / Q000378:metabolism; D004258:DNA Polymerase III / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D004271:DNA, Fungal / Q000096:biosynthesis / Q000235:genetics*; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
chemical_list: D018797:Cell Cycle Proteins; C508610:Csm3p protein, S cerevisiae; D004271:DNA, Fungal; C441579:MRC1 protein, S cerevisiae; C546594:POL30 protein, S cerevisiae; D018809:Proliferating Cell Nuclear Antigen; D029701:Saccharomyces cerevisiae Proteins; D004257:DNA Polymerase II; D004258:DNA Polymerase III