title: Quality control mechanisms exclude incorrect polymerases from the eukaryotic replication fork.
abstract: The eukaryotic genome is primarily replicated by two DNA polymerases, Pol ε and Pol δ, that function on the leading and lagging strands, respectively. Previous studies have established recruitment mechanisms whereby Cdc45-Mcm2-7-GINS (CMG) helicase binds Pol ε and tethers it to the leading strand, and PCNA (proliferating cell nuclear antigen) binds tightly to Pol δ and recruits it to the lagging strand. The current report identifies quality control mechanisms that exclude the improper polymerase from a particular strand. We find that the replication factor C (RFC) clamp loader specifically inhibits Pol ε on the lagging strand, and CMG protects Pol ε against RFC inhibition on the leading strand. Previous studies show that Pol δ is slow and distributive with CMG on the leading strand. However, Saccharomyces cerevisiae Pol δ-PCNA is a rapid and processive enzyme, suggesting that CMG may bind and alter Pol δ activity or position it on the lagging strand. Measurements of polymerase binding to CMG demonstrate Pol ε binds CMG with a Kdvalue of 12 nM, but Pol δ binding CMG is undetectable. Pol δ, like bacterial replicases, undergoes collision release upon completing replication, and we propose Pol δ-PCNA collides with the slower CMG, and in the absence of a stabilizing Pol δ-CMG interaction, the collision release process is triggered, ejecting Pol δ on the leading strand. Hence, by eviction of incorrect polymerases at the fork, the clamp machinery directs quality control on the lagging strand and CMG enforces quality control on the leading strand.
authors.affiliation: Howard Hughes Medical Institute and Rockefeller University, New York, NY 10065.
authors.forename: Grant D
authors.initials: GD
authors.lastname: Schauer
authors.affiliation: Howard Hughes Medical Institute and Rockefeller University, New York, NY 10065 odonnel@rockefeller.edu.
authors.forename: Michael E
authors.initials: ME
authors.lastname: O'Donnell
mesh_terms: D004257:DNA Polymerase II / Q000378:metabolism*; D004258:DNA Polymerase III / Q000378:metabolism*; D004261:DNA Replication / Q000235:genetics*; D005057:Eukaryotic Cells / Q000378:metabolism*; D064110:Minichromosome Maintenance Proteins / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011786:Quality Control; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism; D051818:Replication Protein C / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism
chemical_list: D018809:Proliferating Cell Nuclear Antigen; D029701:Saccharomyces cerevisiae Proteins; D012324:RNA-Dependent RNA Polymerase; D004257:DNA Polymerase II; D004258:DNA Polymerase III; D051818:Replication Protein C; D064110:Minichromosome Maintenance Proteins
keywords: DNA polymerase; PCNA; clamp loader; replication; replisome