Abstract
SummaryThe eukaryotic replisome, organized around the Cdc45-MCM-GINS (CMG) helicase, orchestrates chromosome replication. Multiple factors associate directly with CMG, including Ctf4 and the heterotrimeric fork protection complex (Csm3/Tof1 and Mrc1), which has important roles including aiding normal replication rates and stabilizing stalled forks. How these proteins interface with CMG to execute these functions is poorly understood. Here we present 3 to 3.5 Å resolution electron cryomicroscopy (cryo-EM) structures comprising CMG, Ctf4, and the fork protection complex at a replication fork. The structures provide high-resolution views of CMG-DNA interactions, revealing a mechanism for strand separation, and show Csm3/Tof1 “grip” duplex DNA ahead of CMG via a network of interactions important for efficient replication fork pausing. Although Mrc1 was not resolved in our structures, we determine its topology in the replisome by cross-linking mass spectrometry. Collectively, our work reveals how four highly conserved replisome components collaborate with CMG to facilitate replisome progression and maintain genome stability.
Highlights
Replication of eukaryotic genomes is initiated when double-hexameric minichromosome maintenance (MCM) complexes are activated to form two CMG helicases (Douglas et al, 2018; Yeeles et al, 2015)
Because doublestranded DNA (dsDNA) is bent away from Ctf4 toward Csm3/Tof1 at the front of the replisome, partner proteins that dock onto the replisome via Ctf4 may be located a considerable distance from the parental DNA duplex approaching the fork junction. This may be of particular significance for Pol a, which is involved in parental H3-H4 transfer to the lagging strand in conjunction with the N-terminal extension (NTE) of Mcm2 (Gan et al, 2018) (Figure S4H)
The fork pausing defect displayed by Csm3-5A/Tof1-3A likely represents a specific defect in responding to the replication fork barriers (RFBs) and is not a consequence of the protein being absent from the replisome. These findings demonstrate the Csm3/Tof1 dsDNA grip is required for efficient replication fork pausing at the RFB
Summary
Baretic and Jenkyn-Bedford et al describe cryo-EM structures of the CMG helicase bound to the fork protection complex and Ctf at a replication fork. They reveal the position of Mrc in the replisome, how Csm3/Tof engage CMG and DNA to facilitate fork pausing, and suggest a mechanism for strand separation. June 4, 2020 a 2020 MRC Laboratory of Molecular Biology
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