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A Ctf4 trimer couples the CMG helicase to DNA polymerase alpha in the eukaryotic replisome

DOI: 10.1038/nature13234 DOI Help
PMID: 24805245 PMID Help

Authors: Aline C. Simon (Department of Biochemistry, University of Cambridge, Cambridge) , Jin C. Zhou (Clare Hall Laboratories, Cancer Research UK, London, UK) , Raj Perera (Department of Biochemistry, University of Cambridge, Cambridge) , Frederick Van Deursen (Cancer Research UK Manchester Institute, University of Manchester, Manchester, UK) , Cecile Evrin (MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee , UK) , Marina Ivanova (Cancer Research UK, London Research Institute) , Mairi Kilkenny (Department of Biochemistry, University of Cambridge, Cambridge, U.K.) , Ludovic Renault (Clare Hall Laboratories, Cancer Research UK, London, UK) , Svend Kjaer (Protein purification, Cancer Research UK, London Research Institute, London,U.K.) , Dijana Matak-vinković (Department of Chemistry, University of Cambridge, Cambridge,U.K) , Karim Labib (MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee, UK) , Alessandro Costa (Clare Hall Laboratories, Cancer Research UK, London, UK) , Luca Pellegrini (University of Cambridge, U.K.)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Nature , VOL 510 (7504) , PAGES 293 - 297

State: Published (Approved)
Published: May 2014
Diamond Proposal Number(s): 9537

Open Access Open Access

Abstract: Efficient duplication of the genome requires the concerted action of helicase and DNA polymerases at replication forks1 to avoid stalling of the replication machinery and consequent genomic instability2, 3, 4. In eukaryotes, the physical coupling between helicase and DNA polymerases remains poorly understood. Here we define the molecular mechanism by which the yeast Ctf4 protein links the Cdc45–MCM–GINS (CMG) DNA helicase to DNA polymerase alpha; (Pol alpha) within the replisome. We use X-ray crystallography and electron microscopy to show that Ctf4 self-associates in a constitutive disk-shaped trimer. Trimerization depends on a beta-propeller domain in the carboxy-terminal half of the protein, which is fused to a helical extension that protrudes from one face of the trimeric disk. Critically, Pol alpha; and the CMG helicase share a common mechanism of interaction with Ctf4. We show that the amino-terminal tails of the catalytic subunit of Pol alpha; and the Sld5 subunit of GINS contain a conserved Ctf4-binding motif that docks onto the exposed helical extension of a Ctf4 protomer within the trimer. Accordingly, we demonstrate that one Ctf4 trimer can support binding of up to three partner proteins, including the simultaneous association with both Pol alpha; and GINS. Our findings indicate that Ctf4 can couple two molecules of Pol α to one CMG helicase within the replisome, providing a new model for lagging-strand synthesis in eukaryotes that resembles the emerging model for the simpler replisome of Escherichia coli5, 6, 7, 8. The ability of Ctf4 to act as a platform for multivalent interactions illustrates a mechanism for the concurrent recruitment of factors that act together at the fork.

Journal Keywords: Amino; Catalytic; Conserved; Crystallography; X-Ray; DNA; DNA-Binding; DNA-Directed; Microscopy; Electron; Minichromosome; Models; Molecular; Multienzyme; Nuclear; Protein; Quaternary; Protein; Saccharomyces; Saccharomyces cerevisiae Proteins

Subject Areas: Biology and Bio-materials

Instruments: I03-Macromolecular Crystallography , I24-Microfocus Macromolecular Crystallography