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Structure of the human clamp loader reveals an autoinhibited conformation of a substrate-bound AAA+ switch
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Structure of the human clamp loader reveals an autoinhibited conformation of a substrate-bound AAA+ switch
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Journal Article
Structure of the human clamp loader reveals an autoinhibited conformation of a substrate-bound AAA+ switch
2020
Overview
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.
Publisher
National Academy of Sciences
Subject
/ Adenosine Triphosphate - chemistry
/ Adenosine Triphosphate - metabolism
/ Antigens
/ ATP
/ ATPases Associated with Diverse Cellular Activities - chemistry
/ ATPases Associated with Diverse Cellular Activities - metabolism
/ Binding
/ DNA
/ DNA Replication - physiology
/ Humans
/ Loaders
/ Mutation
/ Proliferating cell nuclear antigen
/ Proliferating Cell Nuclear Antigen - chemistry
/ Proliferating Cell Nuclear Antigen - metabolism
/ Proliferating Cell Nuclear Antigen - ultrastructure
/ Proteins
/ Regulatory mechanisms (biology)
/ Replication Protein C - chemistry
/ Replication Protein C - metabolism
/ Replication Protein C - ultrastructure
/ Sliding
/ Yeast
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