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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.

Background Calcium phosphate-based bone graft substitutes are used to facilitate healing in bony defects caused by trauma or created during surgery. Here, we present an injectable calcium phosphate-based bone void filler that has been purposefully formulated with hyaluronic acid to offer a longer working time for ease of injection into bony defects that are difficult to access during minimally invasive surgery. Methods The bone substitute material deliverability and physical properties were characterized, and in vivo response was evaluated in a critical size distal femur defect in skeletally mature rabbits to 26 weeks. The interface with the host bone, implant degradation, and resorption were assessed with time. Results The calcium phosphate bone substitute material could be injected as a paste within the working time window of 7–18 min, and then self-cured at body temperature within 10 min. The material reached a maximum ultimate compressive strength of 8.20 ± 0.95 MPa, similar to trabecular bone. The material was found to be biocompatible and osteoconductive in vivo out to 26 weeks, with new bone formation and normal bone architecture observed at 6 weeks, as demonstrated by histological evaluation, microcomputed tomography, and radiographic evaluation. Conclusions These findings show that the material properties and performance are well suited for minimally invasive percutaneous delivery applications.

All life requires loading ring-shaped sliding clamp protein complexes onto DNA. The sliding clamp loader is a conserved AAA+ ATPase that binds the sliding clamp, opens the ring, and places it onto DNA. While recent structural work on both the canonical and 'alternative' clamp loaders has shed light into how these machines perform their task once, it remains unclear how clamp loaders are recycled to load multiple sliding clamps. Here, we present structures of the clamp loader Replication Factor C (RFC) in absence of sliding clamp or supplemented nucleotide. Our structures indicate that RFC holds onto ADP tightly in at least two of its four ATPase active sites, suggesting that nucleotide exchange is regulated. Our molecular dynamics simulations and biochemical data indicate that binding of the sliding clamp PCNA causes rapid exchange of tightly bound ADP. Our data suggests that PCNA acts as a nucleotide exchange factor by prying apart adjacent subunits, providing a pathway for ADP release. We propose that, by using its own substrate as a nucleotide exchange factor, RFC excludes off-pathway states that would arise from binding DNA prior to PCNA.

Clamp loaders are pentameric ATPases that place circular sliding clamps onto DNA, where they function in DNA replication and genome integrity. The central activity of a clamp loader is the opening of the ring-shaped sliding clamp, and the subsequent binding to primer-template (p/t)-junctions. The general architecture of clamp loaders is conserved across all life, suggesting that their mechanism is retained. Recent structural studies of the eukaryotic clamp loader Replication Factor C (RFC) revealed that it functions using a crab-claw mechanism, where clamp opening is coupled to a massive conformational change in the loader. Here we investigate the clamp loading mechanism of the clamp loader at high resolution using cryo-electron microscopy (cryo-EM). We find that the clamp loader opens the clamp using a crab-claw motion at a single pivot point, whereas the eukaryotic RFC loader uses motions distributed across the complex. Furthermore, we find clamp opening occurs in multiple steps, starting with a partly open state with a spiral conformation, and proceeding to a wide open clamp in a surprising planar geometry. Finally, our structures in the presence of p/t-junctions illustrate how clamp closes around p/t-junctions and how the clamp loader initiates release from the loaded clamp. Our results reveal mechanistic distinctions in a macromolecular machine that is conserved across all domains of life.

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 actively 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 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 cryo-EM to an overall resolution of ~3.4 Å. The active sites of RFC are fully bound to ATP analogs, which is expected to induce opening of the sliding clamp. However, we observe the complex in a conformation prior to PCNA opening, with the clamp loader ATPase modules forming an over-twisted 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 auto-inhibition. The proposed change from an over-twisted to an active conformation reveals a novel 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. Competing Interest Statement The authors have declared no competing interest. Footnotes * https://www.rcsb.org/structure/6vvo * https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-21405

Eutrophication is a challenge to coastal waters around the globe. In many places, nutrient reductions from land-based sources have not been sufficient to achieve desired water quality improvements. Bivalve shellfish have shown promise as an in-water strategy to complement land-based nutrient management. A local-scale production model was used to estimate oyster (Crassostrea virginica) harvest and bioextraction of nitrogen (N) in Great Bay Piscataqua River Estuary (GBP), New Hampshire, USA, because a system-scale ecological model was not available. Farm-scale N removal results (0.072 metric tons acre⁻¹ year⁻¹) were up-scaled to provide a system-wide removal estimate for current (0.61 metric tons year⁻¹), and potential removal (2.35 metric tons year⁻¹) at maximum possible expansion of licensed aquaculture areas. Restored reef N removal was included to provide a more complete picture. Nitrogen removal through reef sequestration was ~ 3 times that of aquaculture. Estimated reef-associated denitrification, based on previously reported rates, removed 0.19 metric tons N year⁻¹. When all oyster processes (aquaculture and reefs) were included, N removal was 0.33% and 0.54% of incoming N for current and expanded acres, respectively. An avoided cost approach, with wastewater treatment as the alternative management measure, was used to estimate the value of the N removed. The maximum economic value for aquaculture-based removal was $105,000 and $405,000 for current and expanded oyster areas, respectively. Combined aquaculture and reef restoration is suggested to maximize N reduction capacity while limiting use conflicts. Comparison of removal based on per oyster N content suggests much lower removal rates than model results, but model harvest estimates are similar to reported harvest. Though results are specific to GBP, the approach is transferable to estuaries that support bivalve aquaculture but do not have complex system-scale hydrodynamic or ecological models.