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Nature. 2014 Jun 12;510(7504):293-297. doi: 10.1038/nature13234. Epub 2014 May 4.

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

Author information

1
Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, UK.
2
Clare Hall Laboratories, Cancer Research U.K. London Research Institute, London EN6 3LD, UK.
3
Cancer Research U.K. Manchester Institute, University of Manchester, Manchester M20 4BX, UK.
4
MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee DD1 5EH, UK.
5
Protein purification, Cancer Research U.K. London Research Institute, London WC2A 3LY, UK.
6
Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, UK.
#
Contributed equally

Abstract

Efficient duplication of the genome requires the concerted action of helicase and DNA polymerases at replication forks to avoid stalling of the replication machinery and consequent genomic instability. 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 α (Pol α) 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 β-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 α 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 α 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 α 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 coli. 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.

PMID:
24805245
PMCID:
PMC4059944
DOI:
10.1038/nature13234
[Indexed for MEDLINE]
Free PMC Article

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