A stable leading strand polymerase/clamp loader complex is required for normal and perturbed eukaryotic DNA replication

Abstract

The eukaryotic replisome must faithfully replicate DNA and cope with replication fork blocks and stalling, while simultaneously promoting sister chromatid cohesion. Ctf18-RFC is an alternative PCNA loader that links all these processes together by an unknown mechanism. Here, we use integrative structural biology combined with yeast genetics and biochemistry to highlight the specific functions that Ctf18-RFC plays within the leading strand machinery via an interaction with the catalytic domain of DNA Pol ε. We show that a large and unusually flexible interface enables this interaction to occur constitutively throughout the cell cycle and regardless of whether forks are replicating or stalled. We reveal that, by being anchored to the leading strand polymerase, Ctf18-RFC can rapidly signal fork stalling to activate the S phase checkpoint. Moreover, we demonstrate that, independently of checkpoint signaling or chromosome cohesion, Ctf18-RFC functions in parallel to Chl1 and Mrc1 to protect replication forks and cell viability.

Eukaryotic clamp loaders and unloaders in the maintenance of genome stability

“…A recent genome-wide analysis of PCNA occupation on the leading and lagging strands consistently showed that Ctf18-RLC preferentially loads PCNA on the leading strand, and that the RFC complex preferentially loads PCNA on the lagging strand 28 . This study, along with other studies, showed that interaction with Pol ε contributes to the recruitment of Ctf18-RLC to replication forks 28,29 . However, Ctf18 is not essential for bulk DNA replication and cannot substitute for Rfc1 deletion.…”

“…Ctf18 mutants defective with Pol ε-binding were originally reported to be sensitive to hydroxyurea and defective in the replication checkpoint, as shown by reduced Rad53 phosphorylation 63 . On the other hand, two recent reports show that hydroxyurea sensitivity and reduced Rad53 phosphorylation are not observed in either Pol ε-binding-defective dcc1 mutants or Dcc1-binding-defective Pol ε mutants 28,29 . However, the same paper showed that Rad52 foci, markers for recombination, and Psf1 phosphorylation, a marker for late origin firing, both of which are normally suppressed by checkpoint activation, are increased in Pol ε-bindingdefective ctf18 mutants treated with hydroxyurea.…”

“…However, the same paper showed that Rad52 foci, markers for recombination, and Psf1 phosphorylation, a marker for late origin firing, both of which are normally suppressed by checkpoint activation, are increased in Pol ε-bindingdefective ctf18 mutants treated with hydroxyurea. This suggests the importance of Ctf18-RFC recruitment to the leading strand for activation of the replication checkpoint 29 . Human CTF18-RLC interacts with Pol ε via DCC1 26 , but it is unknown whether this interaction is also important for checkpoint activation in human cells.…”

“…Ctf18-RLC interacts with Pol ε via Dcc1 26 , and this interaction contributes to the recruitment of Ctf18-RLC to replication forks 28,29 . Ctf18 mutants defective with Pol ε-binding were originally reported to be sensitive to hydroxyurea and defective in the replication checkpoint, as shown by reduced Rad53 phosphorylation 63 .…”