Conserved interaction of Ctf18‐RFC with DNA polymerase ε is critical for maintenance of genome stability in Saccharomyces cerevisiae

Abstract

Human Ctf18-RFC, a PCNA loader complex, interacts with DNA polymerase ε (Polε) through a structure formed by the Ctf18, Dcc1, and Ctf8 subunits. The C-terminal stretch of Ctf18, which is highly conserved from yeast to human, is necessary to form the Polε-capturing structure. We found that in the budding yeast Saccharomyces cerevisiae, Ctf18, Dcc1, and Ctf8 formed the same structure through the conserved C terminus and interacted specifically with Polε. Thus, the specific interaction of Ctf18-RFC with Polε is a conserved feature between these proteins. A C-terminal deletion mutant of Ctf18 (ctf18 ΔC ) exhibited the same high sensitivity to hydroxyurea as the complete deletion strain (ctf18Δ)or ATPase- Author ManuscriptThis article is protected by copyright. All rights reserved deficient mutant (ctf18 K189A), but was somewhat less sensitive to methyl methanesulfonate than either of them. These phenotypes were also observed in dcc1Δ and ctf8Δ, predicted to be deficient in the interaction with Pol ε . Furthermore, both plasmid-loss and gross chromosomal rearrangement (GCR) rates were elevated in ctf18 ΔC

Structural studies of RFCCtf18 reveal a novel chromatin recruitment role for Dcc1

“…Recent studies have also shown that RFC Ctf18 is able to bind to DNA polymerase epsilon (Pol e) via the catalytic subunit Pol2 [57,60,61]. This interaction requires the intact Ctf18 C -Ctf8-Dcc1 complex which we present in this study.…”

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

“…This suggests that ctf18-RAA dcc1WH3Δ/pol2-5A mutants load sufficient PCNA to support cohesion establishment. Whether this is mediated by recruitment of the mutant Ctf18-RFC near forks via alternative interactions or by some residual and transient binding to Pol2, which can only be removed by complete disruption of the Ctf18-1-8 module (Okimoto et al ., 2016), is not understood.…”

“…The Ctf18-1-8 module comprises a triple-barrel domain (TBD) formed from all three proteins and a winged helix hook (WHH), composed of a series of winged-helix domains in the C-terminus of Dcc1 resembling a hook (Grabarczyk, Silkenat and Kisker, 2018; Wade et al ., 2017). Mutations in Ctf18 that disrupt formation of this Ctf18-1-8 module result in similar but not identical phenotypes as entire deletions of Ctf18 (Okimoto et al ., 2016; García-Rodríguez et al ., 2015), suggesting that this module is critical for at least some of the specialized functions of Ctf18-RFC. It has been shown that the Ctf18-1-8 module binds the N-terminal catalytic domain of Pol2 (García-Rodríguez et al ., 2015; Murakami et al ., 2010), the largest subunit of the leading strand Pol ε, and that this interaction increases the clamp loader activity of Ctf18-RFC (Fujisawa et al ., 2017).…”

Control of Genome Integrity by RFC Complexes; Conductors of PCNA Loading onto and Unloading from Chromatin during DNA Replication

“…Eco1 associates with PCNA and promotes cohesion by acetylating the cohesion subunit Smc3 during S phase [ 84 , 85 , 86 , 87 ]. The fact that loss of either Ctf18, Dcc1, or Ctf8 causes cohesion defects and that these molecules form a DNA polymerase ε binding module suggest that sufficient levels of PCNA on the leading strand must be supplied by Ctf18-RFC, which would support polymerase ε and Eco1 acetylation activity [ 55 , 88 , 89 ].…”