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.
Highlights
Chromosome replication is one of the most complex processes in cell biology and is mediated by an extensive set of proteins, in eukaryotes where DNA synthesis is coupled to a variety of other processes, such as chromatin regeneration, checkpoint signaling, and the establishment of cohesion between sister chromatids
A defining feature of the bacterial replisome is that the clamp loader connects the DnaB helicase to three copies of the DNA polymerase III complex that jointly synthesize the leading and lagging strands
Pol a Cannot Be the Only Factor that Is Linked to the CMG Helicase by the Ctf4 C-Terminal Domain
Summary
Chromosome replication is one of the most complex processes in cell biology and is mediated by an extensive set of proteins, in eukaryotes where DNA synthesis is coupled to a variety of other processes, such as chromatin regeneration, checkpoint signaling, and the establishment of cohesion between sister chromatids. Of the many factors that mediate chromosome duplication, a core assembles around the essential DNA helicase at replication forks to form a dynamic assembly called the replisome (Yao and O’Donnell, 2010). The reasons for replisome assembly are understood poorly in eukaryotes, where replisome structure is ill defined, multiple components are still of unknown function, and in vitro reconstitution of chromosome duplication is still at an early stage (Yeeles et al, 2015). A defining feature of the bacterial replisome is that the clamp loader connects the DnaB helicase to three copies of the DNA polymerase III complex that jointly synthesize the leading and lagging strands. The same principles should apply to the eukaryotic replisome, the underlying molecular mechanisms are very different, as the eubacterial and eukaryotic machineries evolved separately (Georgescu et al, 2015), and the eukaryotic replisome contains many factors not found in its bacterial counterpart
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