CMG–Pol epsilon dynamics suggests a mechanism for the establishment of leading-strand synthesis in the eukaryotic replisome

Abstract

The replisome unwinds and synthesizes DNA for genome duplication. In eukaryotes, the Cdc45-MCM-GINS (CMG) helicase and the leading-strand polymerase, Pol epsilon, form a stable assembly. The mechanism for coupling DNA unwinding with synthesis is starting to be elucidated, however the architecture and dynamics of the replication fork remain only partially understood, preventing a molecular understanding of chromosome replication. To address this issue, we conducted a systematic single-particle EM study on multiple permutations of the reconstituted CMG-Pol epsilon assembly. Pol epsilon contains two flexibly tethered lobes. The noncatalytic lobe is anchored to the motor of the helicase, whereas the polymerization domain extends toward the side of the helicase. We observe two alternate configurations of the DNA synthesis domain in the CMG-bound Pol epsilon. We propose that this conformational switch might control DNA template engagement and release, modulating replisome progression.DNA replication | CMG helicase | DNA polymerase | single-particle electron microscopy D NA replication is catalyzed by the replisome, a molecular machine that coordinates DNA unwinding and synthesis (1). These two functions must be tightly coordinated to prevent the rise of genome instability, which is a major cause of cancer. DNA unwinding by a replicative helicase involves single-strand translocation of a hexameric motor, whereas DNA synthesis requires template priming by a primase and extension by dedicated replicative DNA polymerases (2). In eukaryotes, the helicase function is performed by the Cdc45-MCM-GINS (CMG) complex (3, 4) and the primase function is played by Pol alpha (5), whereas DNA synthesis is catalyzed by two specialized DNA polymerases, Pol epsilon and delta. According to the consensus view, Pol epsilon synthesizes the leading and Pol delta the lagging strand (6-11). However, recent studies indicate that the division of labor between replicative polymerases might be more promiscuous than originally thought (12, 13). In in vitroreconstituted DNA replication reactions, Pol delta can support leading-strand duplication (11, 14), but switching from Pol delta to epsilon is necessary for efficient establishment of leadingstrand synthesis (14). The mechanism of substrate handoff between the two polymerases is currently unknown.Recent breakthroughs in structural biology begin to provide an architectural framework to understand the interaction between helicase and polymerases at the replication fork. For example, studies on the CMG helicase and its subcomplexes have established that the MCM is a six-member ring with an N-terminal domain that serves as a processivity collar (15) and a C-terminal ATPase motor domain that provides the DNA unwinding function (16-21). High-resolution cryo-EM analysis has shown that the ATPase motor translocates on the leading-strand template (22), in agreement with work on Xenopus embryo extracts (23). The GINS and Cdc45 components of the CMG bind to the side and stabilize the N-terminal domain of the...

Noise in the Machine: Alternative Pathway Sampling is the Rule During DNA Replication

“…In line with these findings, Pol δ can take over leading‐strand synthesis in a dysfunctional Pol ϵ background . Additionally, structural evidence has emerged showing two conformations of Pol ϵ in complex with CMG, suggestive of a Pol δ‐Pol ϵ switch on the leading strand . In this model, initial leading‐strand synthesis is uncoupled from CMG unwinding activity.…”

“…The prevailing view in the field is that eukaryotic replication proceeds with continuous synthesis of the leading strand and discontinuous synthesis of the lagging strand. However, a closer look at leading‐strand synthesis also shows discontinuous synthesis, as the leading‐strand polymerase is not always stably attached to DNA . In the past, a variety of studies using biochemical and genetic techniques generated the widely accepted view that Pol ϵ performs leading‐strand synthesis and Pol δ performs lagging‐strand synthesis (reviewed in Ref …”

Regulation and Modulation of Human DNA Polymerase δ Activity and Function

“…These studies show that the catalytic domain of Pol ε is flexibly attached to its noncatalytic domain (which is engaged in complex with the CMG helicase). The catalytic domain adopts two conformations: it is proposed that in one conformation the catalytic domain is actively engaged with the DNA and in the other one it is disengaged [168,169]. In the context of the human replisome, we envisage that encounter with replication blocks stalls Pol ε, leading to the disengagement of the catalytic domain, followed by a switch to Pol δ4/PDIP46 which performs the bypass synthesis (Figure 7).…”

“…The model shown in Figure 7 incorporates recent structural and functional studies of the yeast replisome from the Diffley laboratory [168][169][170][171]. These studies show that the catalytic domain of Pol ε is flexibly attached to its noncatalytic domain (which is engaged in complex with the CMG helicase).…”

“…It was proposed that Pol could also disengage from the DNA during replication  stress [168,169]. Replication stress, broadly defined as encounter with replication barriers due to template lesions or complex DNA structures might be addressed by similar mechanisms to that which are well established in relation to Pol  [88,179,180] and are based on the switching of specialized polymerases such as the TLS polymerases.…”