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

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 instability2-4. 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 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 N-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 paradigm for lagging-strand synthesis in eukaryotes that resembles the emerging model for the simpler replisome of E. coli5-8. 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.

Ctf4-related protein recruits LHP1-PRC2 to maintain H3K27me3 levels in dividing cells in Arabidopsis thaliana

“…Yeast Ctf4 forms a functional trimer that acts as a multivalent interface between the MCM2-7 helicase-associated GINS complex and DNA-polα during replication (29). This interaction is mediated through specific recognition of a peptide motif found in DNA-polα and Sld5 with the α-helical fold of Ctf4 (29,34,35). We confirmed an interaction between plant EOL1 and SLD5 (Fig.…”

“…This domain structure is found in yeast Ctf4 and metazoan Actinodin 1 (AND1)/ WD-and-High-mobility-group-domain protein 1 (WDHD1), components of the nuclear DNA replisome (34,35). A recently solved crystal structure of Ctf4 showed that DUF3639 is part of a novel WD40-related domain that forms a β-propeller with six blades (β-6-prop), forming a stable trimer (29). The carboxyl-terminal end of Ctf4 extends as α-helical fold that can interact with a motif found in both DNA-Polα and Sld5 (29).…”

“…A recently solved crystal structure of Ctf4 showed that DUF3639 is part of a novel WD40-related domain that forms a β-propeller with six blades (β-6-prop), forming a stable trimer (29). The carboxyl-terminal end of Ctf4 extends as α-helical fold that can interact with a motif found in both DNA-Polα and Sld5 (29). Sld5 is part of the GINS complex associated with the replicative MCM2-7 helicase complex, which unwinds the parental DNA double strand.…”

“…Mammalian WDHD1/AND1 features a High Mobility Group (HMG) domain in a carboxyl-terminal extension that is absent from yeast and plant homologs. Yeast Ctf4 forms a functional trimer that acts as a multivalent interface between the MCM2-7 helicase-associated GINS complex and DNA-polα during replication (29). This interaction is mediated through specific recognition of a peptide motif found in DNA-polα and Sld5 with the α-helical fold of Ctf4 (29,34,35).…”

“…We provide evidence that the gene encodes the A. thaliana homolog of yeast Chromosome transmission fidelity 4 (Ctf4), which acts in a trimeric complex to couple DNA-Polα and the DNA helicase complex during DNA replication (29). We show that the function of EOL1 within the PcG pathway depends on H3K27me3 activity and contributes to H3K27me3 inheritance during cell division.…”