Unexpected new insights into DNA clamp loaders: Eukaryotic clamp loaders contain a second DNA site for recessed 5' ends that facilitates repair and signals DNA damage: Eukaryotic clamp loaders contain a second DNA site for recessed 5' ends that facilitates repair and signals DNA damage.

Clamp loaders are pentameric AAA+ assemblies that use ATP to open and close circular DNA sliding clamps around DNA. Clamp loaders show homology in all organisms, from bacteria to human. The eukaryotic PCNA clamp is loaded onto 3' primed DNA by the replication factor C (RFC) hetero-pentameric clamp loader. Eukaryotes also have three alternative RFC-like clamp loaders (RLCs) in which the Rfc1 subunit is substituted by another protein. One of these is the yeast Rad24-RFC (Rad17-RFC in human) that loads a 9-1-1 heterotrimer clamp onto a recessed 5' end of DNA. Recent structural studies of Rad24-RFC have discovered an unexpected 5' DNA binding site on the outside of the clamp loader and reveal how a 5' end can be utilized for loading the 9-1-1 clamp onto DNA. In light of these results, new studies reveal that RFC also contains a 5' DNA binding site, which functions in gap repair. These studies also reveal many new features of clamp loaders. As reviewed herein, these recent studies together have transformed our view of the clamp loader mechanism.

Optimizing CMG helicase and CMG-dependent replication assays by designing DNA fork substrates and choosing nucleotide analogues for helicase preloading.

The replication machinery that synthesizes new copies of chromosomal DNA is located at the junction where double-stranded DNA is separated into its two strands. This replication fork DNA structure is at the heart of most assays involving DNA helicases. The helicase enzyme unwinds the replication fork structure into two single-stranded templates which are converted into two daughter duplexes by other proteins, including DNA polymerases. In eukaryotes, the CMG (Cdc45/Mcm2-7/GINS) helicase plays the pivotal role of unwinding the parental duplex DNA and at the same time interacts with numerous other proteins, including the leading strand polymerase, Pol ɛ. This chapter first describes how we design and prepare synthetic replication forks used in our CMG-related assays. Then we describe how to load CMG onto the fork. The Mcm2-7 motor subunits of CMG form a closed ring, as do all cellular replicative helicases, that encircles ssDNA for helicase function. Thus, the first step in these assays is the loading of CMG onto the fork DNA, followed by DNA unwinding and replication. We explain protocols for different strategies of preloading CMG onto the DNA fork using different ATP analogues. Additionally, the presence of Mcm10, an intimate partner of CMG, affects how CMG is preloaded onto a fork substrate.

Expression of recombinant multi-protein complexes in Saccharomyces cerevisiae.

Baker's yeast, Saccharomyces cerevisiae, is a versatile system for expression of recombinant eukaryotic proteins. This system is simple to use and does not require extraordinary expertise nor tissue culture facilities. Proteins expressed in the yeast system provide eukaryotic post-translational modifications, making it superior to bacterial expression for factors that require post-translational modification. In addition, it is quite simple to co-express multiple genes at the same time, for recombinant production of large multi-protein complexes. In this chapter, we provide protocols for inducible expression of recombinant genes from episomal plasmid vectors, and protocols for integration of the recombinant genes into the chromosomes of yeast, which enables simple rapid growth of expression cells and induction of recombinant protein complexes in non-selectable rich media.

Water skating: How polymerase sliding clamps move on DNA.

Polymerase sliding clamps are ring-shaped proteins that encircle duplex DNA and hold polymerases to DNA for high processivity during synthesis. The crystal structure of clamp-DNA complex reveals that the DNA is highly tilted through the clamp with extensive interaction with the clamp inner surface. In contrast to the tilted clamp-DNA interaction without DNA polymerases, recent structures of replicative polymerases of bacteria, eukaryotes, and archaea that are bound to the clamp and DNA show that the polymerase positions DNA straight through the clamp without direct protein-DNA contacts. Instead, the clamp-to-DNA interaction is mediated by one or two layers of water. Hence, clamps 'water skate' on DNA during function with replicative polymerases from all domains of life, providing a nearly frictionless bearing for fast and processive DNA synthesis.

In vitro Assays for Eukaryotic Leading/Lagging Strand DNA Replication.

The eukaryotic replisome is a multiprotein complex that duplicates DNA. The replisome is sculpted to couple continuous leading strand synthesis with discontinuous lagging strand synthesis, primarily carried out by DNA polymerases ε and δ, respectively, along with helicases, polymerase α-primase, DNA sliding clamps, clamp loaders and many other proteins. We have previously established the mechanisms by which the polymerases ε and δ are targeted to their 'correct' strands, as well as quality control mechanisms that evict polymerases when they associate with an 'incorrect' strand. Here, we provide a practical guide to differentially assay leading and lagging strand replication in vitro using pure proteins.

Mcm10 promotes rapid isomerization of CMG-DNA for replisome bypass of lagging strand DNA blocks.

Replicative helicases in all cell types are hexameric rings that unwind DNA by steric exclusion in which the helicase encircles the tracking strand only and excludes the other strand from the ring. This mode of translocation allows helicases to bypass blocks on the strand that is excluded from the central channel. Unlike other replicative helicases, eukaryotic CMG helicase partially 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 greatly stimulates its helicase activity in vitro. Most significantly, Mcm10 enables CMG and the replisome to bypass blocks on the non-tracking DNA strand. We demonstrate that bypass occurs without displacement of the blocks and therefore Mcm10 must isomerize the CMG-DNA complex to achieve the bypass function.

DNA Replication: How Does a Sliding Clamp Slide?

DNA sliding clamps are rings that tether certain enzymes to DNA. How clamp proteins slide on DNA has remained a mystery. A new crystal structure, together with molecular dynamics and NMR studies, has revealed how the human PCNA clamp slides on DNA.

Action of CMG with strand-specific DNA blocks supports an internal unwinding mode for the eukaryotic replicative helicase.

Replicative helicases are ring-shaped hexamers that encircle DNA for duplex unwinding. The currently accepted view of hexameric helicase function is by steric exclusion, where the helicase encircles one DNA strand and excludes the other, acting as a wedge with an external DNA unwinding point during translocation. Accordingly, strand-specific blocks only affect these helicases when placed on the tracking strand, not the excluded strand. We examined the effect of blocks on the eukaryotic CMG and, contrary to expectations, blocks on either strand inhibit CMG unwinding. A recent cryoEM structure of yeast CMG shows that duplex DNA enters the helicase and unwinding occurs in the central channel. The results of this report inform important aspects of the structure, and we propose that CMG functions by a modified steric exclusion process in which both strands enter the helicase and the duplex unwinding point is internal, followed by exclusion of the non-tracking strand.

Reconstitution of a eukaryotic replisome reveals the mechanism of asymmetric distribution of DNA polymerases.

Eukaryotes require 3 DNA polymerases for normal replisome operations, DNA polymerases (Pol) α, delta and epsilon. Recent biochemical and structural studies support the asymmetric use of these polymerases on the leading and lagging strands. Pol epsilon interacts with the 11-subunit CMG helicase, forming a 15-protein leading strand complex that acts processively in leading strand synthesis in vitro, but Pol epsilon is inactive on the lagging strand. The opposite results are observed for Pol delta with CMG. Pol delta is highly active on the lagging strand in vitro, but has only feeble activity with CMG on the leading strand. Pol α also functions with CMG to prime both strands, and is even capable of extending both strands with CMG present. However, extensive DNA synthesis by Pol α is sharply curtailed by the presence of either Pol epsilon or Pol delta, which limits the role of the low fidelity Pol α to the initial priming of synthesis.

The eukaryotic CMG helicase pumpjack and integration into the replisome.

The eukaryotic replisome is α multiprotein machine that contains DNA polymerases, sliding clamps, helicase, and primase along with several factors that participate in cell cycle and checkpoint control. The detailed structure of the 11-subunit CMG helicase (Cdc45/Mcm2-7/GINS) has been solved recently by cryoEM single-particle 3D reconstruction and reveals pumpjack motions that imply an unexpected mechanism of DNA translocation. CMG is also the organizing center of the replisome. Recent in vitro reconstitution of leading and lagging strand DNA synthesis has enabled structural analysis of the replisome. By building the replisome in stages from pure proteins, single-particle EM studies have identified the overall architecture of the eukaryotic replisome. Suprisingly leading and lagging strand polymerases bind to opposite faces of the CMG helicase, unlike the long-held view that DNA polymerases are located in back of the helicase to act on the unwound strands.

Evolution of replication machines.

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.

The Eukaryotic Replisome Goes Under the Microscope.

The machinery at the eukaryotic replication fork has seen many new structural advances using electron microscopy and crystallography. Recent structures of eukaryotic replisome components include the Mcm2-7 complex, the CMG helicase, DNA polymerases, a Ctf4 trimer hub and the first look at a core replisome of 20 different proteins containing the helicase, primase, leading polymerase and a lagging strand polymerase. The eukaryotic core replisome shows an unanticipated architecture, with one polymerase sitting above the helicase and the other below. Additionally, structures of Mcm2 bound to an H3/H4 tetramer suggest a direct role of the replisome in handling nucleosomes, which are important to DNA organization and gene regulation. This review provides a summary of some of the many recent advances in the structure of the eukaryotic replisome.

Bacterial and Eukaryotic Replisome Machines.

Cellular genomic DNA is replicated by a multiprotein replisome machine. The replisome contains numerous essential factors that unwind, prime and synthesize each of the two strands of duplex DNA. The antiparallel structure of DNA, and unidirectional activity of DNA polymerases, requires the two strands of DNA to be extended in opposite directions, and this structural feature requires distinctive processes for synthesis of the two strands. Genome duplication is of central importance to all cell types, and one may expect the replisome apparatus to be conserved from bacteria to human, as is the case with RNA polymerase driven transcription and ribosome mediated translation. However, it is known that the replication factors of bacteria are not homologous to those of archaea and eukaryotes, indicating that the replication process evolved twice, independently, rather than from a common ancestor cell. Thus, the different domains of life may exhibit significant differences in their mechanistic strategy of replication. In this review, we compare and contrast the different structures and mechanistic features of the cellular replication machinery in the three domains of life.

Reconstitution of a eukaryotic replisome reveals suppression mechanisms that define leading/lagging strand operation.

We have reconstituted a eukaryotic leading/lagging strand replisome comprising 31 distinct polypeptides. This study identifies a process unprecedented in bacterial replisomes. While bacteria and phage simply recruit polymerases to the fork, we find that suppression mechanisms are used to position the distinct eukaryotic polymerases on their respective strands. Hence, Pol ε is active with CMG on the leading strand, but it is unable to function on the lagging strand, even when Pol δ is not present. Conversely, Pol δ-PCNA is the only enzyme capable of extending Okazaki fragments in the presence of Pols ε and α. We have shown earlier that Pol δ-PCNA is suppressed on the leading strand with CMG (Georgescu et al., 2014). We propose that CMG, the 11-subunit helicase, is responsible for one or both of these suppression mechanisms that spatially control polymerase occupancy at the fork.

A proposal: Evolution of PCNA's role as a marker of newly replicated DNA.

Processivity clamps that hold DNA polymerases to DNA for processivity were the first proteins known to encircle the DNA duplex. At the time, polymerase processivity was thought to be the only function of ring shaped processivity clamps. But studies from many laboratories have identified numerous proteins that bind and function with sliding clamps. Among these processes are mismatch repair and nucleosome assembly. Interestingly, there exist polymerases that are highly processive and do not require clamps. Hence, DNA polymerase processivity does not intrinsically require that sliding clamps evolved for this purpose. We propose that polymerases evolved to require clamps as a way of ensuring that clamps are deposited on newly replicated DNA. These clamps are then used on the newly replicated daughter strands, for processes important to genomic integrity, such as mismatch repair and the assembly of nucleosomes to maintain epigenetic states of replicating cells during development.

CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication.

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.

Mechanism of asymmetric polymerase assembly at the eukaryotic replication fork.

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.

Replisome mechanics: lagging strand events that influence speed and processivity.

The antiparallel structure of DNA requires lagging strand synthesis to proceed in the opposite direction of the replication fork. This imposes unique events that occur only on the lagging strand, such as primase binding to DnaB helicase, RNA synthesis, and SS B antigen (SSB) displacement during Okazaki fragment extension. Single-molecule and ensemble techniques are combined to examine the effect of lagging strand events on the Escherichia coli replisome rate and processivity. We find that primase activity lowers replisome processivity but only when lagging strand extension is inoperative. rNTPs also lower replisome processivity. However, the negative effects of primase and rNTPs on processivity are overcome by the extra grip on DNA provided by the lagging strand polymerases. Visualization of single molecules reveals that SSB accumulates at forks and may wrap extensive amounts of single-strand DNA. Interestingly SSB has an inter-strand positive effect on the rate of the leading strand based in its interaction with the replicase χ-subunit. Further, the lagging strand polymerase is faster than leading strand synthesis, indicating that replisome rate is limited by the helicase. Overall, lagging strand events that impart negative effects on the replisome are counterbalanced by the positive effects of SSB and additional sliding clamps during Okazaki fragment extension.