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

Katy Stokes,Alicja Winczura,Boyuan Song,Giacomo De Piccoli,Daniel B Grabarczyk

doi:10.1093/nar/gkaa541

Katy Stokes, Alicja Winczura + Show 3 more

Open Access

https://doi.org/10.1093/nar/gkaa541

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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.

Highlights

The faithful maintenance of genetic information is essential for cell viability and regulated proliferation; strikingly, defects in chromosome duplication and segregation are a powerful source of genomic instability and a hallmark of cell transformation [1]
Our structure reveals that the complete Ctf18-18/Pol2 catalytic domain (Pol2CAT) complex has a large interface (Figure 1B) burying an area of 1363 A 2, which is more than double the 612 A 2 area buried in the minimal complex
Entirely new interactions are formed by the Ctf18-1-8 triple-barrel domain (TBD), which has rotated towards the polymerase (Figure 1C) to interact with Pol2EXO and the Pol2 thumb (Pol2THUMB) subdomain in two discontinuous interaction patches (Supplementary Figure S1B)

Summary

Introduction

The faithful maintenance of genetic information is essential for cell viability and regulated proliferation; strikingly, defects in chromosome duplication and segregation are a powerful source of genomic instability and a hallmark of cell transformation [1]. DNA Pol ε, ␣ and ␦, the first required for the synthesis of the majority the leading strand and the latters for initial DNA synthesis following origin firing and the synthesis of the lagging strand [5,6] Both Pol ε and ␦ are processive DNA polymerases that interact with the PCNA sliding clamp [7]. Deletions of a single component of the replisome often can cause considerable defects in DNA synthesis, in chromosome cohesion [15] and activation of the S phase checkpoint kinase Rad in response to fork stalling [16,17], highlighting how DNA synthesis is closely intertwined with other processes necessary for the maintenance of genome stability. The replicative factor Ctf18–Dcc1–Ctf8–Rfc complex (Ctf18-RFC) exemplifies these overlapping functions at forks

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