The human ATAD5 has evolved unique structural elements to function exclusively as a PCNA unloader.

Humans have three different proliferating cell nuclear antigen (PCNA) clamp-loading complexes: RFC and CTF18-RFC load PCNA onto DNA, but ATAD5-RFC can only unload PCNA from DNA. The underlying structural basis of ATAD5-RFC unloading is unknown. We show here that ATAD5 has two unique locking loops that appear to tie the complex into a rigid structure, and together with a domain that plugs the DNA-binding chamber, prevent conformation changes required for DNA binding, likely explaining why ATAD5-RFC is exclusively a PCNA unloader. These features are conserved in the yeast PCNA unloader Elg1-RFC. We observe intermediates in which PCNA bound to ATAD5-RFC exists as a closed planar ring, a cracked spiral or a gapped spiral. Surprisingly, ATAD5-RFC can open a PCNA gap between PCNA protomers 2 and 3, different from the PCNA protomers 1 and 3 gap observed in all previously characterized clamp loaders.

Cryo-EM reveals a nearly complete PCNA loading process and unique features of the human alternative clamp loader CTF18-RFC.

The DNA sliding clamp PCNA is a multipurpose platform for DNA polymerases and many other proteins involved in DNA metabolism. The topologically closed PCNA ring needs to be cracked open and loaded onto DNA by a clamp loader, e.g., the well-studied pentameric ATPase complex RFC (RFC1-5). The CTF18-RFC complex is an alternative clamp loader found recently to bind the leading strand DNA polymerase ε and load PCNA onto leading strand DNA, but its structure and the loading mechanism have been unknown. By cryo-EM analysis of in vitro assembled human CTF18-RFC-DNA-PCNA complex, we have captured seven loading intermediates, revealing a detailed PCNA loading mechanism onto a 3'-ss/dsDNA junction by CTF18-RFC. Interestingly, the alternative loader has evolved a highly mobile CTF18 AAA+ module likely to lower the loading activity, perhaps to avoid competition with the RFC and to limit its role to leading strand clamp loading. To compensate for the lost stability due to the mobile AAA+ module, CTF18 has evolved a unique ß-hairpin motif that reaches across RFC2 to interact with RFC5, thereby stabilizing the pentameric complex. Further, we found that CTF18 also contains a separation pin to locally melt DNA from the 3'-end of the primer; this ensures its ability to load PCNA to any 3'-ss/dsDNA junction, facilitated by the binding energy of the E-plug to the major groove. Our study reveals unique structural features of the human CTF18-RFC and contributes to a broader understanding of PCNA loading by the alternative clamp loaders.

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.

Smc5/6's multifaceted DNA binding capacities stabilize branched DNA structures.

Smc5/6 is an evolutionarily conserved SMC complex with roles in DNA replication and repair, as well as in viral DNA restriction. Understanding its multiple functions has been hampered by a lack of mechanistic studies on how the Smc5/6 complex associates with different types of DNA. Here we address this question by simultaneously visualizing the behavior of Smc5/6 on three types of DNA, namely double-stranded (ds) DNA, single-stranded (ss) DNA, and junction DNA formed by juxtaposed ss- and dsDNA, using correlative single-molecule fluorescence and force microscopy. We find that Smc5/6 displays distinct behaviors toward different types of DNA, dynamically associating with dsDNA while stably binding to junction DNA. Mechanistically, both the Nse1-3-4 subcomplex and ATP binding enhance the complex's dsDNA association. In contrast, Smc5/6's assembly onto ssDNA emanating from junction DNA, which occurs even in the presence high-affinity ssDNA binders, is aided by Nse1-3-4, but not by ATP. Moreover, we show that Smc5/6 protects junction DNA stability by preventing ssDNA annealing. The multifaceted DNA association behaviors of Smc5/6 provide a framework for understanding its diverse functions in genome maintenance and viral DNA restriction.

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.

Origin recognition complex harbors an intrinsic nucleosome remodeling activity.

Eukaryotic DNA replication is initiated at multiple chromosomal sites known as origins of replication that are specifically recognized by the origin recognition complex (ORC) containing multiple ATPase sites. In budding yeast, ORC binds to specific DNA sequences known as autonomously replicating sequences (ARSs) that are mostly nucleosome depleted. However, nucleosomes may still inhibit the licensing of some origins by occluding ORC binding and subsequent MCM helicase loading. Using purified proteins and single-molecule visualization, we find here that the ORC can eject histones from a nucleosome in an ATP-dependent manner. The ORC selectively evicts H2A-H2B dimers but leaves the (H3-H4)2 tetramer on DNA. It also discriminates canonical H2A from the H2A.Z variant, evicting the former while retaining the latter. Finally, the bromo-adjacent homology (BAH) domain of the Orc1 subunit is essential for ORC-mediated histone eviction. These findings suggest that the ORC is a bona fide nucleosome remodeler that functions to create a local chromatin environment optimal for origin activity.

Nucleosome-directed replication origin licensing independent of a consensus DNA sequence.

The numerous enzymes and cofactors involved in eukaryotic DNA replication are conserved from yeast to human, and the budding yeast Saccharomyces cerevisiae (S.c.) has been a useful model organism for these studies. However, there is a gap in our knowledge of why replication origins in higher eukaryotes do not use a consensus DNA sequence as found in S.c. Using in vitro reconstitution and single-molecule visualization, we show here that S.c. origin recognition complex (ORC) stably binds nucleosomes and that ORC-nucleosome complexes have the intrinsic ability to load the replicative helicase MCM double hexamers onto adjacent nucleosome-free DNA regardless of sequence. Furthermore, we find that Xenopus laevis nucleosomes can substitute for yeast ones in engaging with ORC. Combined with re-analyses of genome-wide ORC binding data, our results lead us to propose that the yeast origin recognition machinery contains the cryptic capacity to bind nucleosomes near a nucleosome-free region and license origins, and that this nucleosome-directed origin licensing paradigm generalizes to all eukaryotes.

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.

CMG helicase can use ATPγS to unwind DNA: Implications for the rate-limiting step in the reaction mechanism.

The adenosine triphosphate (ATP) analog ATPγS often greatly slows or prevents enzymatic ATP hydrolysis. The eukaryotic CMG (Cdc45, Mcm2 to 7, GINS) replicative helicase is presumed unable to hydrolyze ATPγS and thus unable to perform DNA unwinding, as documented for certain other helicases. Consequently, ATPγS is often used to "preload" CMG onto forked DNA substrates without unwinding before adding ATP to initiate helicase activity. We find here that CMG does hydrolyze ATPγS and couples it to DNA unwinding. Indeed, the rate of unwinding of a 20- and 30-mer duplex fork of different sequences by CMG is only reduced 1- to 1.5-fold using ATPγS compared with ATP. These findings imply that a conformational change is the rate-limiting step during CMG unwinding, not hydrolysis. Instead of using ATPγS for loading CMG onto DNA, we demonstrate here that nonhydrolyzable adenylyl-imidodiphosphate (AMP-PNP) can be used to preload CMG onto a forked DNA substrate without unwinding.

Allosteric communication in DNA polymerase clamp loaders relies on a critical hydrogen-bonded junction.

Clamp loaders are AAA+ ATPases that load sliding clamps onto DNA. We mapped the mutational sensitivity of the T4 bacteriophage sliding clamp and clamp loader by deep mutagenesis, and found that residues not involved in catalysis or binding display remarkable tolerance to mutation. An exception is a glutamine residue in the AAA+ module (Gln 118) that is not located at a catalytic or interfacial site. Gln 118 forms a hydrogen-bonded junction in a helical unit that we term the central coupler, because it connects the catalytic centers to DNA and the sliding clamp. A suppressor mutation indicates that hydrogen bonding in the junction is important, and molecular dynamics simulations reveal that it maintains rigidity in the central coupler. The glutamine-mediated junction is preserved in diverse AAA+ ATPases, suggesting that a connected network of hydrogen bonds that links ATP molecules is an essential aspect of allosteric communication in these proteins.

The DNA Replication Machine: Structure and Dynamic Function.

In all cell types, a multi-protein machinery is required to accurately duplicate the large duplex DNA genome. This central life process requires five core replisome factors in all cellular life forms studied thus far. Unexpectedly, three of the five core replisome factors have no common ancestor between bacteria and eukaryotes. Accordingly, the replisome machines of bacteria and eukaryotes have important distinctions in the way that they are organized and function. This chapter outlines the major replication proteins that perform DNA duplication at replication forks, with particular attention to differences and similarities in the strategies used by eukaryotes and bacteria.

Anatomy of a twin DNA replication factory.

The replication of DNA in chromosomes is initiated at sequences called origins at which two replisome machines are assembled at replication forks that move in opposite directions. Interestingly, in vivo studies observe that the two replication forks remain fastened together, often referred to as a replication factory. Replication factories containing two replisomes are well documented in cellular studies of bacteria (Escherichia coli and Bacillus subtilis) and the eukaryote, Saccharomyces cerevisiae. This basic twin replisome factory architecture may also be preserved in higher eukaryotes. Despite many years of documenting the existence of replication factories, the molecular details of how the two replisome machines are tethered together has been completely unknown in any organism. Recent structural studies shed new light on the architecture of a eukaryote replisome factory, which brings with it a new twist on how a replication factory may function.

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.

Replisome bypass of a protein-based R-loop block by Pif1.

Efficient and faithful replication of the genome is essential to maintain genome stability. Replication is carried out by a multiprotein complex called the replisome, which encounters numerous obstacles to its progression. Failure to bypass these obstacles results in genome instability and may facilitate errors leading to disease. Cells use accessory helicases that help the replisome bypass difficult barriers. All eukaryotes contain the accessory helicase Pif1, which tracks in a 5'-3' direction on single-stranded DNA and plays a role in genome maintenance processes. Here, we reveal a previously unknown role for Pif1 in replication barrier bypass. We use an in vitro reconstituted Saccharomyces cerevisiae replisome to demonstrate that Pif1 enables the replisome to bypass an inactive (i.e., dead) Cas9 (dCas9) R-loop barrier. Interestingly, dCas9 R-loops targeted to either strand are bypassed with similar efficiency. Furthermore, we employed a single-molecule fluorescence visualization technique to show that Pif1 facilitates this bypass by enabling the simultaneous removal of the dCas9 protein and the R-loop. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks.