Structure of the human RAD17-RFC clamp loader and 9-1-1 checkpoint clamp bound to a dsDNA-ssDNA junction
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ABSTRACT:
SUBMITTER: Matthew Day
PROVIDER: EMPIAR-11062 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:The RAD9-RAD1-HUS1 (9-1-1) clamp forms one half of the DNA damage checkpoint system that signals the presence of substantial regions of single-stranded DNA arising from replication fork collapse or resection of DNA double strand breaks. Loaded at the 5'-recessed end of a dsDNA-ssDNA junction by the RAD17-RFC clamp loader complex, the phosphorylated C-terminal tail of the RAD9 subunit of 9-1-1 engages with the mediator scaffold TOPBP1 which in turn activates the ATR kinase, localised through the interaction of its constitutive partner ATRIP with RPA-coated ssDNA. Using cryogenic electron microscopy (cryoEM) we have determined the structure of a complex of the human RAD17-RFC clamp loader bound to human 9-1-1, engaged with a dsDNA-ssDNA junction. The structure answers the key questions of how RAD17 confers specificity for 9-1-1 over PCNA, and how the clamp loader specifically recognises the recessed 5' DNA end and fixes the orientation of 9-1-1 on the ssDNA.
| S-EPMC9371934 | biostudies-literature
Project description:The eukaryotic RFC clamp loader couples the energy of ATP hydrolysis to open and close the circular PCNA sliding clamp onto primed sites for use by DNA polymerases and repair factors. Structural studies reveal clamp loaders to be heteropentamers. Each subunit contains a region of homology to AAA+ proteins that defines two domains. The AAA+ domains form a right-handed spiral upon binding ATP. This spiral arrangement generates a DNA binding site within the center of RFC. DNA enters the central chamber through a gap between the AAA+ domains of two subunits. Specificity for a primed template junction is achieved by a third domain that blocks DNA, forcing it to bend sharply. Thus only DNA with a flexible joint can bind the central chamber. DNA entry also requires a slot in the PCNA clamp, which is opened upon binding the AAA+ domains of the clamp loader. ATP hydrolysis enables clamp closing and ejection of RFC, completing the clamp loading reaction.
| S-EPMC3711244 | biostudies-literature
Project description:While telomeres must provide mechanisms to prevent DNA repair and DNA damage checkpoint factors from fusing chromosome ends and causing permanent cell cycle arrest, these factors associate with functional telomeres and play critical roles in the maintenance of telomeres. Previous studies have established that Tel1 (ATM) and Rad3 (ATR) kinases play redundant but essential roles for telomere maintenance in fission yeast. In addition, the Rad9-Rad1-Hus1 (911) and Rad17-RFC complexes work downstream of Rad3 (ATR) in fission yeast telomere maintenance. Here, we investigated how 911, Rad17-RFC and another RFC-like complex Ctf18-RFC contribute to telomere maintenance in fission yeast cells lacking Tel1 and carrying a novel hypomorphic allele of rad3 (DBD-rad3), generated by the fusion between the DNA binding domain (DBD) of the fission yeast telomere capping protein Pot1 and Rad3. Our investigations have uncovered a surprising redundancy for Rad9 and Hus1 in allowing Rad1 to contribute to telomere maintenance in DBD-rad3 tel1 cells. In addition, we found that Rad17-RFC and Ctf18-RFC carry out redundant telomere maintenance functions in DBD-rad3 tel1 cells. Since checkpoint sensor proteins are highly conserved, genetic redundancies uncovered here may be relevant to telomere maintenance and detection of DNA damage in other eukaryotes.
| S-EPMC3133598 | biostudies-literature
Project description:The alternative proliferating-cell nuclear antigen (PCNA)-loader CTF18-RFC forms a stable complex with DNA polymerase ε (Polε). We observed that, under near-physiological conditions, CTF18-RFC alone loaded PCNA inefficiently, but loaded it efficiently when complexed with Polε. During efficient PCNA loading, CTF18-RFC and Polε assembled at a 3΄ primer-template junction cooperatively, and directed PCNA to the loading site. Site-specific photo-crosslinking of directly interacting proteins at the primer-template junction showed similar cooperative binding, in which the catalytic N-terminal portion of Polε acted as the major docking protein. In the PCNA-loading intermediate with ATPγS, binding of CTF18 to the DNA structures increased, suggesting transient access of CTF18-RFC to the primer terminus. Polε placed in DNA synthesis mode using a substrate DNA with a deoxidised 3΄ primer end did not stimulate PCNA loading, suggesting that DNA synthesis and PCNA loading are mutually exclusive at the 3΄ primer-template junction. Furthermore, PCNA and CTF18-RFC-Polε complex engaged in stable trimeric assembly on the template DNA and synthesised DNA efficiently. Thus, CTF18-RFC appears to be involved in leading-strand DNA synthesis through its interaction with Polε, and can load PCNA onto DNA when Polε is not in DNA synthesis mode to restore DNA synthesis.
| S-EPMC5416766 | biostudies-literature
Project description:Replication-factor C (RFC) is a protein complex that loads the processivity clamp PCNA onto DNA. Elg1 is a conserved protein with homology to the largest subunit of RFC, but its function remained enigmatic. Here, we show that yeast Elg1 interacts physically and genetically with PCNA, in a manner that depends on PCNA modification, and exhibits preferential affinity for SUMOylated PCNA. This interaction is mediated by three small ubiquitin-like modifier (SUMO)-interacting motifs and a PCNA-interacting protein box close to the N-terminus of Elg1. These motifs are important for the ability of Elg1 to maintain genomic stability. SUMOylated PCNA is known to recruit the helicase Srs2, and in the absence of Elg1, Srs2 and SUMOylated PCNA accumulate on chromatin. Strains carrying mutations in both ELG1 and SRS2 exhibit a synthetic fitness defect that depends on PCNA modification. Our results underscore the importance of Elg1, Srs2 and SUMOylated PCNA in the maintenance of genomic stability.
| S-EPMC2928695 | biostudies-literature
Project description:Replication factor C (RFC) is a five-subunit complex that loads proliferating cell nuclear antigen (PCNA) clamps onto primer-template DNA (ptDNA) during replication. RFC subunits belong to the AAA(+) superfamily, and their ATPase activity drives interactions between the clamp loader, the clamp, and the ptDNA, leading to topologically linked PCNA·ptDNA. We report the kinetics of transient events in Saccharomyces cerevisiae RFC-catalyzed PCNA loading, including ATP-induced RFC activation, PCNA opening, ptDNA binding, ATP hydrolysis, PCNA closing, and PCNA·ptDNA release. This detailed perspective enables assessment of individual RFC-A, RFC-B, RFC-C, RFC-D, and RFC-E subunit functions in the reaction mechanism. Functions have been ascribed to RFC subunits previously based on a steady-state analysis of 'arginine-finger' ATPase mutants; however, pre-steady-state analysis provides a different view. The central subunit RFC-C serves as a critical swivel point in the clamp loader. ATP binding to this subunit initiates RFC activation, and the clamp loader adopts a spiral conformation that stabilizes PCNA in a corresponding open spiral. The importance of RFC subunit response to ATP binding decreases as RFC-C>RFC-D>RFC-B, with RFC-A being unnecessary. RFC-C-dependent activation of RFC also enables ptDNA binding, leading to the formation of the RFC·ATP·PCNA(open)·ptDNA complex. Subsequent ATP hydrolysis leads to complex dissociation, with RFC-D activity contributing the most to rapid ptDNA release. The pivotal role of the RFC-B/C/D subunit ATPase core in clamp loading is consistent with the similar central location of all three ATPase active subunits of the Escherichia coli clamp loader.
| S-EPMC3269524 | biostudies-literature
Project description:DNA replication requires the sliding clamp, a ring-shaped protein complex that encircles DNA, where it acts as an essential cofactor for DNA polymerases and other proteins. The sliding clamp needs to be opened and installed onto DNA by a clamp loader ATPase of the AAA+ family. The human clamp loader replication factor C (RFC) and sliding clamp proliferating cell nuclear antigen (PCNA) are both essential and play critical roles in several diseases. Despite decades of study, no structure of human RFC has been resolved. Here, we report the structure of human RFC bound to PCNA by cryogenic electron microscopy to an overall resolution of ∼3.4 Å. The active sites of RFC are fully bound to adenosine 5'-triphosphate (ATP) analogs, which is expected to induce opening of the sliding clamp. However, we observe the complex in a conformation before PCNA opening, with the clamp loader ATPase modules forming an overtwisted spiral that is incapable of binding DNA or hydrolyzing ATP. The autoinhibited conformation observed here has many similarities to a previous yeast RFC:PCNA crystal structure, suggesting that eukaryotic clamp loaders adopt a similar autoinhibited state early on in clamp loading. Our results point to a "limited change/induced fit" mechanism in which the clamp first opens, followed by DNA binding, inducing opening of the loader to release autoinhibition. The proposed change from an overtwisted to an active conformation reveals an additional regulatory mechanism for AAA+ ATPases. Finally, our structural analysis of disease mutations leads to a mechanistic explanation for the role of RFC in human health.
| S-EPMC7519235 | biostudies-literature
Project description:The RAD9-RAD1-HUS1 complex (9-1-1) is a eukaryotic DNA clamp with a crucial role at checkpoints for DNA damage. The ring-like structure of 9-1-1 is opened for loading onto 5' recessed DNA by the clamp loader RAD17 RFC-like complex (RAD17-RLC), in which the RAD17 subunit is responsible for specificity to 9-1-1. Loading of 9-1-1 is required for activation of the ATR-CHK1 checkpoint pathway and the activation is stimulated by a 9-1-1 interacting protein, RHINO, which interacts with 9-1-1 via a recently identified RAD1-binding motif. This discovery led to the hypothesis that other interacting proteins may contain a RAD1-binding motif as well. Here, we show that vertebrate RAD17 proteins also have a putative RAD1-binding motif in their N-terminal regions, and we report the crystal structure of human 9-1-1 bound to a human RAD17 peptide incorporating the motif at 2.1 Å resolution. Our structure confirms that the N-terminal region of RAD17 binds to the RAD1 subunit of 9-1-1 via specific interactions. Furthermore, we show that the RAD1-binding motif of RHINO disturbs the interaction of the N-terminal region of RAD17 with 9-1-1. Our results provide deeper understanding of how RAD17-RLC specifically recognizes 9-1-1 and imply that RHINO has a functional role in 9-1-1 loading/unloading and checkpoint activation.
| S-EPMC10060742 | biostudies-literature
Project description:Cellular replicases contain multiprotein ATPases that load sliding clamp processivity factors onto DNA. We reveal an additional role for the DnaX clamp loader: chaperoning of the replicative polymerase onto a clamp newly bound to DNA. We show that chaperoning confers distinct advantages, including marked acceleration of initiation complex formation. We reveal a requirement for the tau form of DnaX complex to relieve inhibition by single-stranded DNA binding protein during initiation complex formation. We propose that, after loading beta(2), DnaX complex preserves an SSB-free segment of DNA immediately downstream of the primer terminus and chaperones Pol III into that position, preventing competition by SSB. The C-terminal tail of SSB stimulates reactions catalyzed by tau-containing DnaX complexes through a contact distinct from the contact involving the chi subunit. Chaperoning of Pol III by the DnaX complex provides a molecular explanation for how initiation complexes form when supported by the nonhydrolyzed analog ATPgammaS.
| S-EPMC2830912 | biostudies-literature
Project description:The cellular pathways involved in maintaining genome stability halt cell cycle progression in the presence of DNA damage or incomplete replication. Proteins required for this pathway include Rad17, Rad9, Hus1, Rad1, and Rfc-2, Rfc-3, Rfc-4, and Rfc-5. The heteropentamer replication factor C (RFC) loads during DNA replication the homotrimer proliferating cell nuclear antigen (PCNA) polymerase clamp onto DNA. Sequence similarities suggest the biochemical functions of an RSR (Rad17-Rfc2-Rfc3-Rfc4-Rfc5) complex and an RHR heterotrimer (Rad1-Hus1-Rad9) may be similar to that of RFC and PCNA, respectively. RSR purified from human cells loads RHR onto DNA in an ATP-, replication protein A-, and DNA structure-dependent manner. Interestingly, RSR and RFC differed in their ATPase activities and displayed distinct DNA substrate specificities. RSR preferred DNA substrates possessing 5' recessed ends whereas RFC preferred 3' recessed end DNA substrates. Characterization of the biochemical loading reaction executed by the checkpoint clamp loader RSR suggests new insights into the mechanisms underlying recognition of damage-induced DNA structures and signaling to cell cycle controls. The observation that RSR loads its clamp onto a 5' recessed end supports a potential role for RHR and RSR in diverse DNA metabolism, such as stalled DNA replication forks, recombination-linked DNA repair, and telomere maintenance, among other processes.
| S-EPMC261875 | biostudies-literature