How the Eukaryotic Replisome Achieves Rapid and Efficient DNA Replication

Abstract

SummaryThe eukaryotic replisome is a molecular machine that coordinates the Cdc45-MCM-GINS (CMG) replicative DNA helicase with DNA polymerases α, δ, and ε and other proteins to copy the leading- and lagging-strand templates at rates between 1 and 2 kb min−1. We have now reconstituted this sophisticated machine with purified proteins, beginning with regulated CMG assembly and activation. We show that replisome-associated factors Mrc1 and Csm3/Tof1 are crucial for in vivo rates of replisome progression. Additionally, maximal rates only occur when DNA polymerase ε catalyzes leading-strand synthesis together with its processivity factor PCNA. DNA polymerase δ can support leading-strand synthesis, but at slower rates. DNA polymerase δ is required for lagging-strand synthesis, but surprisingly also plays a role in establishing leading-strand synthesis, before DNA polymerase ε engagement. We propose that switching between these DNA polymerases also contributes to leading-strand synthesis under conditions of replicative stress.

Human mismatch repair system balances mutation rates between strands by removing more mismatches from the lagging strand

“…Another recent study suggests that Pol delta can replicate both DNA strands near replication origins (Yeeles et al 2017). We believe that these results cannot affect our inferences, as they suggest that Pol delta replicates both strands only at replication origins, while the bulk of the genome is replicated according to the classical model.…”

“…Studies in yeast indicate that the effectiveness of each of these steps depends on the mismatch type and that MMR compensates for the infidelity of polymerases (Kunkel 2011;St Charles et al 2015). The classical model of the eukaryotic replication fork (Larrea et al 2010) suggests a division of labor in replication of the leading and lagging strands among the major DNA polymerases, with polymerase epsilon (Pol epsilon) replicating the leading strand and polymerases alpha (Pol alpha) and delta (Pol delta) replicating the lagging strand, with the possible exception of replication origins and other specific regions where Pol delta may contribute to replication of both strands (Yeeles et al 2017). Under this model, the asymmetry in mutation rates between the leading and the lagging DNA strands may arise due to differences in fidelity of polymerases replicating these strands.…”

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.…”