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

Abstract

SummaryReplisome 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.

Regulation and Modulation of Human DNA Polymerase δ Activity and Function

“…ChIP analysis with antibody against the p125 subunit showed that PDIP46 was present together with two components of the mammalian replisome [56]. These are Mcm2, a component of the CMG (Cdc45-MCM-GINS) helicase [151], and Ctf4, which associates with CMG [152,153]. Thus, the ChIP data supports the idea that PDIP46 is associated with chromatin at or near the replisome.…”

From structure to mechanism—understanding initiation of DNA replication

“…3B; Sun et al 2015). Ctf4 is a homotrimer that acts as a hub of protein interactions, linking several factors involved in DNA synthesis, chromatin remodelling, rDNA stability, DNA recombination, and sister chromatid cohesion to the CMG (Simon et al 2014;Villa et al 2016), while polymerase α primes DNA synthesis prior to extension by processive DNA polymerases (Zhang and O'Donnell 2016). These recent structural findings start to reveal a picture of the eukaryotic replisome assembly process as a whole; namely, the N-terminal MCM2-7 face has a major role in DH organization and stability, helicase activation, controlled opening and closing of the Mcm2/5 gate, and lagging strand DNA synthesis (Fig.…”

Architecture of the Saccharomyces cerevisiae Replisome

“…However, most proteins of the RPC have nearly unknown functions. The Ctf4 trimer recognizes a peptide sequence to bind Pol α and Sld5 suggesting that Ctf4 acts as a platform for recruitment and exchange of different replication factors (Villa et al 2016), in loose analogy to the many proteins that traffic on and off PCNA (Georgescu et al 2015a). The Mrc1 checkpoint mediator is a stably attached replisome component that appears to aid normal replisome progression in addition to its checkpoint function (Lou et al 2008), but how Mrc1 facilitates these activities and the spatial orientation of Mrc1 within the replisome are unknown.…”