Structure of the PCNA unloader Elg1-RFC.

During DNA replication, the proliferating cell nuclear antigen (PCNA) clamps are loaded onto primed sites for each Okazaki fragment synthesis by the AAA+ heteropentamer replication factor C (RFC). PCNA encircling duplex DNA is quite stable and is removed from DNA by the dedicated clamp unloader Elg1-RFC. Here, we show the cryo-EM structure of Elg1-RFC in various states with PCNA. The structures reveal essential features of Elg1-RFC that explain how it is dedicated to PCNA unloading. Specifically, Elg1 contains two external loops that block opening of the Elg1-RFC complex for DNA binding, and an "Elg1 plug" domain that fills the central DNA binding chamber, thereby reinforcing the exclusive PCNA unloading activity of Elg1-RFC. Elg1-RFC was capable of unloading PCNA using non-hydrolyzable AMP-PNP. Both RFC and Elg1-RFC could remove PCNA from covalently closed circular DNA, indicating that PCNA unloading occurs by a mechanism that is distinct from PCNA loading. Implications for the PCNA unloading mechanism are discussed.

Mechanism of eukaryotic origin unwinding is a dual helicase DNA shearing process.

DNA replication in all cells begins with the melting of base pairs at the duplex origin to allow access to single-stranded DNA templates which are replicated by DNA polymerases. In bacteria, origin DNA is presumed to be melted by accessory proteins that allow loading of two ring-shaped replicative helicases around single-strand DNA (ssDNA) for bidirectional unwinding and DNA replication. In eukaryotes, by contrast, two replicative CMG (Cdc45-Mcm2-7-GINS) helicases are initially loaded head to head around origin double-strand DNA (dsDNA), and there does not appear to be a separate origin unwinding factor. This led us to investigate whether head-to-head CMGs use their adenosine triphosphate (ATP)-driven motors to initiate duplex DNA unwinding at the origin. Here, we show that CMG tracks on one strand of the duplex while surrounding it, and this feature allows two head-to-head CMGs to unwind dsDNA by using their respective motors to pull on opposite strands of the duplex. We further show that while CMG is capable of limited duplex unwinding on its own, the extent of unwinding is greatly and rapidly stimulated by addition of the multifunctional CMG-binding protein Mcm10 that is critical for productive initiation of DNA replication in vivo. On the basis of these findings, we propose that Mcm10 is a processivity or positioning factor that helps translate the work performed by the dual CMG motors at the origin into productive unwinding that facilitates bidirectional DNA replication.

Structures of 9-1-1 DNA checkpoint clamp loading at gaps from start to finish and ramification on biology.

Rad24-RFC (replication factor C) loads the 9-1-1 checkpoint clamp onto the recessed 5' ends by binding a 5' DNA at an external surface site and threading the 3' single-stranded DNA (ssDNA) into 9-1-1. We find here that Rad24-RFC loads 9-1-1 onto DNA gaps in preference to a recessed 5' end, thus presumably leaving 9-1-1 on duplex 3' ss/double-stranded DNA (dsDNA) after Rad24-RFC ejects from DNA. We captured five Rad24-RFC-9-1-1 loading intermediates using a 10-nt gap DNA. We also determined the structure of Rad24-RFC-9-1-1 using a 5-nt gap DNA. The structures reveal that Rad24-RFC is unable to melt DNA ends and that a Rad24 loop limits the dsDNA length in the chamber. These observations explain Rad24-RFC's preference for a preexisting gap of over 5-nt ssDNA and suggest a direct role of the 9-1-1 in gap repair with various TLS (trans-lesion synthesis) polymerases in addition to signaling the ATR kinase.

Molecular choreography of primer synthesis by the eukaryotic Pol α-primase.

The eukaryotic polymerase α (Pol α) synthesizes an RNA-DNA hybrid primer of 20-30 nucleotides. Pol α is composed of Pol1, Pol12, Primase 1 (Pri1), and Pri2. Pol1 and Pri1 contain the DNA polymerase and RNA primase activities, respectively. It has been unclear how Pol α hands over an RNA primer from Pri1 to Pol1 for DNA primer extension, and how the primer length is defined. Here we report the cryo-EM analysis of yeast Pol α in the apo, primer initiation, primer elongation, RNA primer hand-off from Pri1 to Pol1, and DNA extension states, revealing a series of very large movements. We reveal a critical point at which Pol1-core moves to take over the 3'-end of the RNA from Pri1. DNA extension is limited by a spiral motion of Pol1-core. Since both Pri1 and Pol1-core are flexibly attached to a stable platform, primer growth produces stress that limits the primer length.

Molecular choreography of primer synthesis by the eukaryotic Pol α-primase.

The eukaryotic polymerase α (Pol α) is a dual-function DNA polymerase/primase complex that synthesizes an RNA-DNA hybrid primer of 20-30 nucleotides for DNA replication. Pol α is composed of Pol1, Pol12, Primase 1 (Pri1), and Pri2, with Pol1 and Pri1 containing the DNA polymerase activity and RNA primase activity, respectively, whereas Pol12 and Pri2 serve a structural role. It has been unclear how Pol α hands over an RNA primer made by Pri1 to Pol1 for DNA primer extension, and how the primer length is defined, perhaps due to the difficulty in studying the highly mobile structure. Here we report a comprehensive cryo-EM analysis of the intact 4-subunit yeast Pol α in the apo, primer initiation, primer elongation, RNA primer hand-off from Pri1 to Pol1, and DNA extension states in a 3.5 Å - 5.6 Å resolution range. We found that Pol α is a three-lobed flexible structure. Pri2 functions as a flexible hinge that holds together the catalytic Pol1-core, and the noncatalytic Pol1 CTD that binds to Pol 12 to form a stable platform upon which the other components are organized. In the apo state, Pol1-core is sequestered on the Pol12-Pol1-CTD platform, and Pri1 is mobile perhaps in search of a template. Upon binding a ssDNA template, a large conformation change is induced that enables Pri1 to perform RNA synthesis, and positions Pol1-core to accept the future RNA primed site 50 Å upstream of where Pri1 binds. We reveal in detail the critical point at which Pol1-core takes over the 3'-end of the RNA from Pri1. DNA primer extension appears limited by the spiral motion of Pol1-core while Pri2-CTD stably holds onto the 5' end of the RNA primer. Since both Pri1 and Pol1-core are attached via two linkers to the platform, primer growth will produce stress within this "two-point" attachment that may limit the length of the RNA-DNA hybrid primer. Hence, this study reveals the large and dynamic series of movements that Pol α undergoes to synthesize a primer for DNA replication.

Structures of 9-1-1 DNA checkpoint clamp loading at gaps from start to finish and ramification to biology.

Recent structural studies show the Rad24-RFC loads the 9-1-1 checkpoint clamp onto a recessed 5' end by binding the 5' DNA on Rad24 at an external surface site and threading the 3' ssDNA into the well-established internal chamber and into 9-1-1. We find here that Rad24-RFC loads 9-1-1 onto DNA gaps in preference to a recessed 5' DNA end, thus presumably leaving 9-1-1 on a 3' ss/ds DNA after Rad24-RFC ejects from the 5' gap end and may explain reports of 9-1-1 directly functioning in DNA repair with various TLS polymerases, in addition to signaling the ATR kinase. To gain a deeper understanding of 9-1-1 loading at gaps we report high-resolution structures of Rad24-RFC during loading of 9-1-1 onto 10-nt and 5-nt gapped DNAs. At a 10-nt gap we captured five Rad24-RFC-9-1-1 loading intermediates in which the 9-1-1 DNA entry gate varies from fully open to fully closed around DNA using ATPγS, supporting the emerging view that ATP hydrolysis is not needed for clamp opening/closing, but instead for dissociation of the loader from the clamp encircling DNA. The structure of Rad24-RFC-9-1-1 at a 5-nt gap shows a 180° axially rotated 3'-dsDNA which orients the template strand to bridge the 3'- and 5'- junctions with a minimum 5-nt ssDNA. The structures reveal a unique loop on Rad24 that limits the length of dsDNA in the inner chamber, and inability to melt DNA ends unlike RFC, thereby explaining Rad24-RFC's preference for a preexisting ssDNA gap and suggesting a direct role in gap repair in addition to its checkpoint role.

SV40 T-antigen uses a DNA shearing mechanism to initiate origin unwinding.

Duplication of DNA genomes requires unwinding of the double-strand (ds) DNA so that each single strand (ss) can be copied by a DNA polymerase. The genomes of eukaryotic cells are unwound by two ring-shaped hexameric helicases that initially encircle dsDNA but transition to ssDNA for function as replicative helicases. How the duplex is initially unwound, and the role of the two helicases in this process, is poorly understood. We recently described an initiation mechanism for eukaryotes in which the two helicases are directed inward toward one another and shear the duplex open by pulling on opposite strands of the duplex while encircling dsDNA [L. D. Langston, M. E. O'Donnell, eLife 8, e46515 (2019)]. Two head-to-head T-Antigen helicases are long known to be loaded at the SV40 origin. We show here that T-Antigen tracks head (N-tier) first on ssDNA, opposite the direction proposed for decades. We also find that SV40 T-Antigen tracks directionally while encircling dsDNA and mainly tracks on one strand of the duplex in the same orientation as during ssDNA translocation. Further, two inward directed T-Antigen helicases on dsDNA are able to melt a 150-bp duplex. These findings explain the "rabbit ear" DNA loops observed at the SV40 origin by electron microscopy and reconfigure how the DNA loops emerge from the double hexamer relative to earlier models. Thus, the mechanism of DNA shearing by two opposing helicases is conserved in a eukaryotic viral helicase and may be widely used to initiate origin unwinding of dsDNA genomes.

Cryo-EM structures reveal that RFC recognizes both the 3'- and 5'-DNA ends to load PCNA onto gaps for DNA repair.

RFC uses ATP to assemble PCNA onto primed sites for replicative DNA polymerases δ and ε. The RFC pentamer forms a central chamber that binds 3' ss/ds DNA junctions to load PCNA onto DNA during replication. We show here five structures that identify a second DNA binding site in RFC that binds a 5' duplex. This 5' DNA site is located between the N-terminal BRCT domain and AAA+ module of the large Rfc1 subunit. Our structures reveal ideal binding to a 7-nt gap, which includes 2 bp unwound by the clamp loader. Biochemical studies show enhanced binding to 5 and 10 nt gaps, consistent with the structural results. Because both 3' and 5' ends are present at a ssDNA gap, we propose that the 5' site facilitates RFC's PCNA loading activity at a DNA damage-induced gap to recruit gap-filling polymerases. These findings are consistent with genetic studies showing that base excision repair of gaps greater than 1 base requires PCNA and involves the 5' DNA binding domain of Rfc1. We further observe that a 5' end facilitates PCNA loading at an RPA coated 30-nt gap, suggesting a potential role of the RFC 5'-DNA site in lagging strand DNA synthesis.

DNA is loaded through the 9-1-1 DNA checkpoint clamp in the opposite direction of the PCNA clamp.

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.

Structure of eukaryotic DNA polymerase δ bound to the PCNA clamp while encircling DNA.

The DNA polymerase (Pol) δ of Saccharomyces cerevisiae (S.c.) is composed of the catalytic subunit Pol3 along with two regulatory subunits, Pol31 and Pol32. Pol δ binds to proliferating cell nuclear antigen (PCNA) and functions in genome replication, repair, and recombination. Unique among DNA polymerases, the Pol3 catalytic subunit contains a 4Fe-4S cluster that may sense the cellular redox state. Here we report the 3.2-Šcryo-EM structure of S.c. Pol δ in complex with primed DNA, an incoming ddTTP, and the PCNA clamp. Unexpectedly, Pol δ binds only one subunit of the PCNA trimer. This singular yet extensive interaction holds DNA such that the 2-nm-wide DNA threads through the center of the 3-nm interior channel of the clamp without directly contacting the protein. Thus, a water-mediated clamp and DNA interface enables the PCNA clamp to "waterskate" along the duplex with minimum drag. Pol31 and Pol32 are positioned off to the side of the catalytic Pol3-PCNA-DNA axis. We show here that Pol31-Pol32 binds single-stranded DNA that we propose underlies polymerase recycling during lagging strand synthesis, in analogy to Escherichia coli replicase. Interestingly, the 4Fe-4S cluster in the C-terminal CysB domain of Pol3 forms the central interface to Pol31-Pol32, and this strategic location may explain the regulation of the oxidation state on Pol δ activity, possibly useful during cellular oxidative stress. Importantly, human cancer and other disease mutations map to nearly every domain of Pol3, suggesting that all aspects of Pol δ replication are important to human health and disease.

Structure of the polymerase ε holoenzyme and atomic model of the leading strand replisome.

The eukaryotic leading strand DNA polymerase (Pol) ε contains 4 subunits, Pol2, Dpb2, Dpb3 and Dpb4. Pol2 is a fusion of two B-family Pols; the N-terminal Pol module is catalytic and the C-terminal Pol module is non-catalytic. Despite extensive efforts, there is no atomic structure for Pol ε holoenzyme, critical to understanding how DNA synthesis is coordinated with unwinding and the DNA path through the CMG helicase-Pol ε-PCNA clamp. We show here a 3.5-Šcryo-EM structure of yeast Pol ε revealing that the Dpb3-Dpb4 subunits bridge the two DNA Pol modules of Pol2, holding them rigid. This information enabled an atomic model of the leading strand replisome. Interestingly, the model suggests that an OB fold in Dbp2 directs leading ssDNA from CMG to the Pol ε active site. These results complete the DNA path from entry of parental DNA into CMG to exit of daughter DNA from PCNA.

DNA unwinding mechanism of a eukaryotic replicative CMG helicase.

High-resolution structures have not been reported for replicative helicases at a replication fork at atomic resolution, a prerequisite to understanding the unwinding mechanism. The eukaryotic replicative CMG (Cdc45, Mcm2-7, GINS) helicase contains a Mcm2-7 motor ring, with the N-tier ring in front and the C-tier motor ring behind. The N-tier ring is structurally divided into a zinc finger (ZF) sub-ring followed by the oligosaccharide/oligonucleotide-binding (OB) fold ring. Here we report the cryo-EM structure of CMG on forked DNA at 3.9 Å, revealing that parental DNA enters the ZF sub-ring and strand separation occurs at the bottom of the ZF sub-ring, where the lagging strand is blocked and diverted sideways by OB hairpin-loops of Mcm3, Mcm4, Mcm6, and Mcm7. Thus, instead of employing a specific steric exclusion process, or even a separation pin, unwinding is achieved via a "dam-and-diversion tunnel" mechanism that does not require specific protein-DNA interaction. The C-tier motor ring contains spirally configured PS1 and H2I loops of Mcms 2, 3, 5, 6 that translocate on the spirally-configured leading strand, and thereby pull the preceding DNA segment through the diversion tunnel for strand separation.

Ctf4 organizes sister replisomes and Pol α into a replication factory.

The current view is that eukaryotic replisomes are independent. Here we show that Ctf4 tightly dimerizes CMG helicase, with an extensive interface involving Psf2, Cdc45, and Sld5. Interestingly, Ctf4 binds only one Pol α-primase. Thus, Ctf4 may have evolved as a trimer to organize two helicases and one Pol α-primase into a replication factory. In the 2CMG-Ctf43-1Pol α-primase factory model, the two CMGs nearly face each other, placing the two lagging strands toward the center and two leading strands out the sides. The single Pol α-primase is centrally located and may prime both sister replisomes. The Ctf4-coupled-sister replisome model is consistent with cellular microscopy studies revealing two sister forks of an origin remain attached and are pushed forward from a protein platform. The replication factory model may facilitate parental nucleosome transfer during replication.

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.

Structure of eukaryotic CMG helicase at a replication fork and implications to replisome architecture and origin initiation.

The eukaryotic CMG (Cdc45, Mcm2-7, GINS) helicase consists of the Mcm2-7 hexameric ring along with five accessory factors. The Mcm2-7 heterohexamer, like other hexameric helicases, is shaped like a ring with two tiers, an N-tier ring composed of the N-terminal domains, and a C-tier of C-terminal domains; the C-tier contains the motor. In principle, either tier could translocate ahead of the other during movement on DNA. We have used cryo-EM single-particle 3D reconstruction to solve the structure of CMG in complex with a DNA fork. The duplex stem penetrates into the central channel of the N-tier and the unwound leading single-strand DNA traverses the channel through the N-tier into the C-tier motor, 5'-3' through CMG. Therefore, the N-tier ring is pushed ahead by the C-tier ring during CMG translocation, opposite the currently accepted polarity. The polarity of the N-tier ahead of the C-tier places the leading Pol ε below CMG and Pol α-primase at the top of CMG at the replication fork. Surprisingly, the new N-tier to C-tier polarity of translocation reveals an unforeseen quality-control mechanism at the origin. Thus, upon assembly of head-to-head CMGs that encircle double-stranded DNA at the origin, the two CMGs must pass one another to leave the origin and both must remodel onto opposite strands of single-stranded DNA to do so. We propose that head-to-head motors may generate energy that underlies initial melting at the origin.

Architecture of the Saccharomyces cerevisiae Replisome.

Eukaryotic replication proteins are highly conserved, and thus study of Saccharomyces cerevisiae replication can inform about this central process in higher eukaryotes including humans. The S. cerevisiae replisome is a large and dynamic assembly comprised of ~50 proteins. The core of the replisome is composed of 31 different proteins including the 11-subunit CMG helicase; RFC clamp loader pentamer; PCNA clamp; the heteroligomeric DNA polymerases ε, δ, and α-primase; and the RPA heterotrimeric single strand binding protein. Many additional protein factors either travel with or transiently associate with these replisome proteins at particular times during replication. In this chapter, we summarize several recent structural studies on the S. cerevisiae replisome and its subassemblies using single particle electron microscopy and X-ray crystallography. These recent structural studies have outlined the overall architecture of a core replisome subassembly and shed new light on the mechanism of eukaryotic replication.

Structure of the eukaryotic replicative CMG helicase suggests a pumpjack motion for translocation.

The CMG helicase is composed of Cdc45, Mcm2-7 and GINS. Here we report the structure of the Saccharomyces cerevisiae CMG, determined by cryo-EM at a resolution of 3.7-4.8 Å. The structure reveals that GINS and Cdc45 scaffold the N tier of the helicase while enabling motion of the AAA+ C tier. CMG exists in two alternating conformations, compact and extended, thus suggesting that the helicase moves like an inchworm. The N-terminal regions of Mcm2-7, braced by Cdc45-GINS, form a rigid platform upon which the AAA+ C domains make longitudinal motions, nodding up and down like an oil-rig pumpjack attached to a stable platform. The Mcm ring is remodeled in CMG relative to the inactive Mcm2-7 double hexamer. The Mcm5 winged-helix domain is inserted into the central channel, thus blocking entry of double-stranded DNA and supporting a steric-exclusion DNA-unwinding model.

The architecture of a eukaryotic replisome.

At the eukaryotic DNA replication fork, it is widely believed that the Cdc45-Mcm2-7-GINS (CMG) helicase is positioned in front to unwind DNA and that DNA polymerases trail behind the helicase. Here we used single-particle EM to directly image a Saccharomyces cerevisiae replisome. Contrary to expectations, the leading strand Pol ɛ is positioned ahead of CMG helicase, whereas Ctf4 and the lagging-strand polymerase (Pol) α-primase are behind the helicase. This unexpected architecture indicates that the leading-strand DNA travels a long distance before reaching Pol ɛ, first threading through the Mcm2-7 ring and then making a U-turn at the bottom and reaching Pol ɛ at the top of CMG. Our work reveals an unexpected configuration of the eukaryotic replisome, suggests possible reasons for this architecture and provides a basis for further structural and biochemical replisome studies.

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.