Ctf4 Is a Hub in the Eukaryotic Replisome that Links Multiple CIP-Box Proteins to the CMG Helicase

Replisome assembly at eukaryotic replication forks connects the DNA helicase to DNA polymerases and many other factors. The helicase binds the leading-strand polymerase directly, but is connected to the Pol α lagging-strand polymerase by the trimeric adaptor Ctf4. Here, we identify new Ctf4 partners in addition to Pol α and helicase, all of which contain a “Ctf4-interacting-peptide” or CIP-box. Crystallographic analysis classifies CIP-boxes into two related groups that target different sites on Ctf4. Mutations in the CIP-box motifs of the Dna2 nuclease or the rDNA-associated protein Tof2 do not perturb DNA synthesis genome-wide, but instead lead to a dramatic shortening of chromosome 12 that contains the large array of rDNA repeats. Our data reveal unexpected complexity of Ctf4 function, as a hub that connects multiple accessory factors to the replisome. Most strikingly, Ctf4-dependent recruitment of CIP-box proteins couples other processes to DNA synthesis, including rDNA copy-number regulation. [Display omitted] •Ctf4 is a hub that links factors with diverse functions to the eukaryotic replisome•Multiple Ctf4 partners bind via short sequences called “CIP-boxes”•The CIP-boxes of Dna2 and Tof2 bind to distinct sites on Ctf4•Interaction of Dna2 and Tof2 with Ctf4 is important for rDNA copy number maintenance Villa et al. identify Ctf4 as a key hub in the eukaryotic replisome. Rather than simply linking DNA helicase to DNA polymerase, Ctf4 couples the replication machinery to multiple factors that contribute to diverse aspects of chromosome duplication.