Structural studies of RFC^Ctf18 reveal a novel chromatin recruitment role for Dcc1

Abstract

Replication factor C complexes load and unload processivity clamps from DNA and are involved in multiple DNA replication and repair pathways. The RFCC tf18 variant complex is required for activation of the intra‐S‐phase checkpoint at stalled replication forks and aids the establishment of sister chromatid cohesion. Unlike other RFC complexes, RFCC tf18 contains two non‐Rfc subunits, Dcc1 and Ctf8. Here, we present the crystal structure of the Dcc1‐Ctf8 heterodimer bound to the C‐terminus of Ctf18. We find that the C‐terminus of Dcc1 contains three‐winged helix domains, which bind to both ssDNA and dsDNA. We further show that these domains are required for full recruitment of the complex to chromatin, and correct activation of the replication checkpoint. These findings provide the first structural data on a eukaryotic seven‐subunit clamp loader and define a new biochemical activity for Dcc1.

Ctf18-RFC and DNA Pol ϵ form a stable leading strand polymerase/clamp loader complex required for normal and perturbed DNA replication

“…We show this is achieved through an unusual protein–protein mechanism that utilizes flexible electrostatic interfaces to enable high binding plasticity. These low-specificity electrostatic patches explain why it was previously thought that the Ctf18-1-8 complex binds to DNA ( 27 , 28 ).…”

“…In an EMSA supershift assay, wild-type Ctf18-1-8 saturated Pol2 CAT -DNA at the lowest tested concentration of 125 nM (Figure 2B ). Ctf18-1-8 alone weakly binds DNA ( 27 , 28 ), but control experiments showed that this is not the cause of any supershifted bands ( Supplementary Figure S1F ). The Dcc1-3A and Ctf18-RAA variants showed a clear binding defect.…”

“…This module is separated from the RFC catalytic module by a large predicted unstructured linker (Figure 1A ). 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 ( 27 , 28 ). Mutations in Ctf18 that disrupt formation of this Ctf18-1-8 module result in similar but not identical phenotypes as entire deletions of Ctf18 ( 29 , 30 ), suggesting that this module is critical for at least some of the specialized functions of Ctf18-RFC.…”

“…It was recently shown that the last winged helix domain, WH3, of Dcc1 assumes a dual function: it can transiently interact with either DNA or a minimal construct of Pol2 ( 27 ). However, despite a triple amino acid mutation within the WH3 domain that completely abolishes both interactions in vitro , surprisingly, deletion of WH3 in vivo only causes a small loss of Ctf18-RFC from replicating forks, while a cohesion defect is only seen when both WH2 and WH3 are deleted ( 28 ). Thus, it remains unclear what the Ctf18-1-8 module is targeting Ctf18-RFC to and under which conditions this occurs.…”

DNA Replication and Sister Chromatid Cohesion 1 (DSCC1) of the Replication Factor Complex CTF18-RFC is Critical for Colon Cancer Cell Growth

“…The prototypical member (Rfc1-RFC) is essential for all replication and repair processes, whereas Rad24-RFC is involved in the DNA-damage checkpoint response 8-10, and Elg1-RFC is involved in proliferating cell nuclear antigen (PCNA) unloading 11 and genomic stability 12-14. CTF18-RFC was originally identified in screens for chromosome mis‐segregation 15-17 in budding yeast ( Saccharomyces cerevisiae ) and is important for the establishment of sister-chromatid cohesion 16, 18 and the activation of the replication-stress checkpoint 15, 19, 20. During DNA-replication processes, CTF18-RFC assists DNA replication by loading the PCNA clamp onto the DNA 21-23, but the depletion of CTF18-RFC reduces the speed of replication-fork traversal 24 and results in defects in telomere metabolism 25 and DNA-repair processes 26, 27.…”