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The 9-1-1 DNA checkpoint clamp is loaded onto 5′-recessed DNA to activate the DNA damage checkpoint that arrests the cell cycle. The 9-1-1 clamp is a heterotrimeric ring that is loaded in Saccharomyces cerevisiae by Rad24-RFC (hRAD17-RFC), an alternate clamp loader in which Rad24 replaces Rfc1 in the RFC1-5 clamp loader of proliferating cell nuclear antigen (PCNA). The 9-1-1 clamp loading mechanism has been a mystery, because, unlike RFC, which loads PCNA onto a 3′-recessed junction, Rad24-RFC loads the 9-1-1 ring onto a 5′-recessed DNA junction. Here we report two cryo-EM structures of Rad24-RFC–DNA with a closed or 27-Å open 9-1-1 clamp. The structures reveal a completely unexpected mechanism by which a clamp can be loaded onto DNA. Unlike RFC, which encircles DNA, Rad24 binds 5′-DNA on its surface, not inside the loader, and threads the 3′ ssDNA overhang into the 9-1-1 clamp from above the ring.

The machines that decode and regulate genetic information require the translation, transcription and replication pathways essential to all living cells. Thus, it might be expected that all cells share the same basic machinery for these pathways that were inherited from the primordial ancestor cell from which they evolved. A clear example of this is found in the translation machinery that converts RNA sequence to protein. The translation process requires numerous structural and catalytic RNAs and proteins, the central factors of which are homologous in all three domains of life, bacteria, archaea and eukarya. Likewise, the central actor in transcription, RNA polymerase, shows homology among the catalytic subunits in bacteria, archaea and eukarya. In contrast, while some “gears” of the genome replication machinery are homologous in all domains of life, most components of the replication machine appear to be unrelated between bacteria and those of archaea and eukarya. This review will compare and contrast the central proteins of the “replisome” machines that duplicate DNA in bacteria, archaea and eukarya, with an eye to understanding the issues surrounding the evolution of the DNA replication apparatus.

Replicative helicases of all cell types are rings that unwind DNA by steric exclusion in which the helicase ring only encircles the tracking strand, excluding the other strand outside the ring. Steric exclusion mediated unwinding enables helicase rings to bypass blocks on the strand that is excluded from the central channel. Unlike other replicative helicases, eukaryotic CMG encircles duplex DNA at a forked junction and is stopped by a block on the non-tracking (lagging) strand. This report demonstrates that Mcm10, an essential replication protein unique to eukaryotes, binds CMG and enables the replisome to bypass blocks on the non-tracking strand, implying that Mcm10 isomerizes the CMG-DNA complex to position only one strand through the central channel. A similar CMG-DNA isomerization is needed at the origin for head-to-head CMGs to bypass one another during formation of bidirectional replication forks.

Sliding clamps encircle DNA and tether polymerases and other factors to the genomic template. However, the molecular mechanism of clamp sliding on DNA is unknown. Using crystallography, NMR and molecular dynamics simulations, here we show that the human clamp PCNA recognizes DNA through a double patch of basic residues within the ring channel, arranged in a right-hand spiral that matches the pitch of B-DNA. We propose that PCNA slides by tracking the DNA backbone via a ‘cogwheel' mechanism based on short-lived polar interactions, which keep the orientation of the clamp invariant relative to DNA. Mutation of residues at the PCNA–DNA interface has been shown to impair the initiation of DNA synthesis by polymerase δ (pol δ). Therefore, our findings suggest that a clamp correctly oriented on DNA is necessary for the assembly of a replication-competent PCNA-pol δ holoenzyme., DNA sliding clamps are ring-shaped proteins that encircle DNA and harbour polymerases and other factors that promote processive DNA replication. Here the authors use X-ray crystallography, NMR and MD simulations to propose a model for a PCNA sliding mechanism that relies on short-lived polar interactions.

DNA replication in eukaryotes is asymmetric, with separate DNA polymerases (Pol) dedicated to bulk synthesis of the leading and lagging strands. Pol α/primase initiates primers on both strands that are extended by Pol ε on the leading strand and by Pol δ on the lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the leading strand and is proposed to recruit Pol ε for leading-strand synthesis, but to date a direct interaction between CMG and Pol ε has not been demonstrated. While purifying CMG helicase overexpressed in yeast, we detected a functional complex between CMG and native Pol ε. Using pure CMG and Pol ε, we reconstituted a stable 15-subunit CMG-Pol ε complex and showed that it is a functional polymerase-helicase on a model replication fork in vitro. On its own, the Pol2 catalytic subunit of Pol ε is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol ε, known to bind the Psf1 protein subunit of CMG, allows stable synthesis with CMG. Dpb2 does not affect Pol δ function with CMG, and thus we propose that the connection between Dpb2 and CMG helps to stabilize Pol ε on the leading strand as part of a 15-subunit leading-strand holoenzyme we refer to as CMGE. Direct binding between Pol ε and CMG provides an explanation for specific targeting of Pol ε to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes.

Eukaryotes use distinct polymerases for leading- and lagging-strand replication, but how they target their respective strands is uncertain. We reconstituted Saccharomyces cerevisiae replication forks and found that CMG helicase selects polymerase (Pol) ɛ to the exclusion of Pol δ on the leading strand. Even if Pol δ assembles on the leading strand, Pol ɛ rapidly replaces it. Pol δ-PCNA is distributive with CMG, in contrast to its high stability on primed ssDNA. Hence CMG will not stabilize Pol δ, instead leaving the leading strand accessible for Pol ɛ and stabilizing Pol ɛ. Comparison of Pol ɛ and Pol δ on a lagging-strand model DNA reveals the opposite. Pol δ dominates over excess Pol ɛ on PCNA-primed ssDNA. Thus, PCNA strongly favors Pol δ over Pol ɛ on the lagging strand, but CMG over-rides and flips this balance in favor of Pol ɛ on the leading strand.

Replicative polymerases are tethered to DNA by sliding clamps for processive DNA synthesis. Despite attachment to a sliding clamp, the polymerase on the lagging strand must cycle on and off DNA for each Okazaki fragment. In the 'collision release' model, the lagging strand polymerase collides with the 5' terminus of an earlier completed fragment, which triggers it to release from DNA and from the clamp. This report examines the mechanism of collision release by the Escherichia coli Pol III polymerase. We find that collision with a 5' terminus does not trigger polymerase release. Instead, the loss of ssDNA on filling in a fragment triggers polymerase to release from the clamp and DNA. Two ssDNA-binding elements are involved, the tau subunit of the clamp loader complex and an OB domain within the DNA polymerase itself. The tau subunit acts as a switch to enhance polymerase binding at a primed site but not at a nick. The OB domain acts as a sensor that regulates the affinity of Pol III to the clamp in the presence of ssDNA.

The process of chromosome duplication faces many obstacles. One way to circumvent blocks is to hop over them by placing a new clamp on a downstream primer. This resembles lagging strand synthesis, where the tight grip of polymerase to the clamp and DNA must be overcome upon completing each Okazaki fragment so it can transfer to new primed sites. This review focuses on recent single-molecule studies showing that Escherichia coli Pol III can hop from one clamp to another without leaving the replication fork. This capability provides a means to circumvent obstacles like transcription or DNA lesions without fork collapse.

Replisomes are dynamic multiprotein machines capable of simultaneously replicating both strands of the DNA duplex. This review focuses on the structure and function of the E. coli replisome, many features of which generalize to other bacteria and eukaryotic cells. For example, the bacterial replisome utilizes clamps and clamp loaders to coordinate the actions required of the trombone model of lagging strand synthesis made famous by Bruce Alberts. All cells contain clamps and clamp loaders and this review summarizes their structure and function. Clamp loaders are pentameric spirals that bind DNA in a structure specific fashion and thread it through the ring shaped clamp. The recent structure of the E. coli beta clamp in complex with primed DNA has implications for how multiple polymerases function on sliding clamps and how the primed DNA template is exchanged between them. Recent studies reveal a remarkable fluidity in replisome function that enables it to bypass template lesions on either DNA strand. During these processes the polymerases within the replisome functionally uncouple from one another. Mechanistic processes that underlie these actions may involve DNA looping, similar to the trombone loops that mediate the lagging strand Okazaki fragment synthesis cycle.

The eukaryotic replication factor C (RFC) clamp loader is an AAA+ spiral-shaped heteropentamer that opens and closes the circular proliferating cell nuclear antigen (PCNA) clamp processivity factor on DNA. In this study, we examined the roles of individual RFC subunits in opening the PCNA clamp. Interestingly, Rfc1, which occupies the position analogous to the delta clamp-opening subunit in the Escherichia coli clamp loader, is not required to open PCNA. The Rfc5 subunit is required to open PCNA. Consistent with this result, Rfc2.3.4.5 and Rfc2.5 subassemblies are capable of opening and unloading PCNA from circular DNA. Rfc5 is positioned opposite the PCNA interface from Rfc1, and therefore, its action with Rfc2 in opening PCNA indicates that PCNA is opened from the opposite side of the interface that the E. coli delta wrench acts upon. This marks a significant departure in the mechanism of eukaryotic and prokaryotic clamp loaders. Interestingly, the Rad.RFC DNA damage checkpoint clamp loader unloads PCNA clamps from DNA. We propose that Rad.RFC may clear PCNA from DNA to facilitate shutdown of replication in the face of DNA damage.