Structure of the human clamp loader reveals an autoinhibited conformation of a substrate-bound AAA+ switch Christl Gaubitza,1, Xingchen Liua,b,1, Joseph Magrinoa,b, Nicholas P. Stonea, Jacob Landecka,b, Mark Hedglinc, and Brian A. Kelcha,2 aDepartment of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester MA 01605; bGraduate School of Biomedical Sciences, University of Massachusetts Medical School, Worcester MA 01605; and cDepartment of Chemistry, The Pennsylvania State University, University Park, PA 16802 Edited by Michael E. O’Donnell, HHMI and Rockefeller University, New York, NY, and approved July 27, 2020 (received for review April 20, 2020) DNA replication requires the sliding clamp, a ring-shaped protein areflexia syndrome (15), Hutchinson–Gilford progeria syn- complex that encircles DNA, where it acts as an essential cofactor drome (16), and in the replication of some viruses (17–19). It for DNA polymerases and other proteins. The sliding clamp needs is unknown whether loading by RFC contributes to PARD to be opened and installed onto DNA by a clamp loader ATPase of disease. the AAA+ family. The human clamp loader replication factor C Clamp loaders are members of the AAA+ family of ATPases (RFC) and sliding clamp proliferating cell nuclear antigen (PCNA) (ATPases associated with various cellular activities), a large are both essential and play critical roles in several diseases. De- protein family that uses the chemical energy of adenosine 5′- spite decades of study, no structure of human RFC has been re- triphosphate (ATP) to generate mechanical force (20). Most solved. Here, we report the structure of human RFC bound to AAA+ proteins form hexameric motors that use an undulating PCNA by cryogenic electron microscopy to an overall resolution ∼ spiral staircase mechanism to processively translocate a substrate of 3.4 Å. The active sites of RFC are fully bound to adenosine – 5′-triphosphate (ATP) analogs, which is expected to induce open- through the motor pore (21 23). Unlike most other AAA+ ing of the sliding clamp. However, we observe the complex in a proteins, clamp loaders do not use ATP hydrolysis as a force- BIOCHEMISTRY conformation before PCNA opening, with the clamp loader ATPase generation step. Instead, the ATP-bound clamp loader forces the modules forming an overtwisted spiral that is incapable of binding sliding clamp ring to open through binding energy alone (24–26). DNA or hydrolyzing ATP. The autoinhibited conformation ob- Subsequent binding of primer–template DNA into the central served here has many similarities to a previous yeast RFC:PCNA chamber of the clamp loader activates ATP hydrolysis, which crystal structure, suggesting that eukaryotic clamp loaders adopt a results in clamp closure and ejection of the clamp loader similar autoinhibited state early on in clamp loading. Our results (27–33). The sliding clamp is now loaded at a primer–template point to a “limited change/induced fit” mechanism in which the junction for use by DNA metabolic enzymes, such as DNA clamp first opens, followed by DNA binding, inducing opening of polymerases. Thus, the clamp loader is an ATP-dependent the loader to release autoinhibition. The proposed change from an protein-remodeling switch (31). overtwisted to an active conformation reveals an additional reg- ulatory mechanism for AAA+ ATPases. Finally, our structural anal- Significance ysis of disease mutations leads to a mechanistic explanation for the role of RFC in human health. DNA replication and repair depend on AAA+ ATPase protein sliding clamp | DNA replication | AAA+ | ATPase | clamp loader complexes called clamp loaders that open and load ring-shaped sliding clamps onto DNA. Using cryogenic electron microscopy, we determined the first structure of the human clamp loader NA replication in all cellular life requires sliding clamps, (RFC), which is in an autoinhibited conformation while bound Dring-shaped protein complexes that encircle DNA to topo- to the sliding clamp (PCNA). We assign this to be a reaction logically link numerous factors to DNA. Sliding clamps are intermediate prior to clamp opening and propose a confor- necessary for DNA synthesis, because they increase polymerase – mational change necessary for activation, leading to a unique processivity and speed by orders of magnitude (1 5). Sliding paradigm for the AAA+ ATPase mechanism. We examined clamps additionally bind and facilitate the function of many RFC’s interaction with a PCNA disease variant, which illumi- other proteins involved in diverse DNA transactions, such as nates how this variant maintains tight interactions with some DNA repair, recombination, and chromatin structure (6). The partners. Finally, mapping of cancer mutations onto RFC’s sliding clamp of eukaryotes, proliferating cell nuclear antigen ’ structure suggests stability as a key factor in proper function (PCNA), is critical for human health. PCNA s central role in and human health. controlling many cancer pathways makes it a common cancer marker (7). Recently, the genetic disease PCNA-associated DNA Author contributions: C.G., X.L., and B.A.K. designed research; C.G., X.L., J.M., N.P.S., J.L., repair disorder (PARD) was shown to be caused by a hypomor- M.H., and B.A.K. performed research; M.H. contributed new reagents/analytic tools; C.G., phic mutation in PCNA that disrupts partner binding (8, 9). X.L., J.M., N.P.S., J.L., M.H., and B.A.K. analyzed data; and C.G., X.L., and B.A.K. wrote PCNA’s ring shape necessitates active loading onto DNA by the paper. the replication factor C (RFC) sliding clamp loader. Clamp The authors declare no competing interest. loaders are pentameric ATPase machines that can open the This article is a PNAS Direct Submission. sliding clamp and close it around DNA. Clamp loaders are found Published under the PNAS license. in all life, although their composition varies across different 1C.G. and X.L. contributed equally to this work. kingdoms (10). The primary clamp loader in eukaryotes con- 2To whom correspondence may be addressed. Email: [email protected]. sists of five distinct proteins, RFC1–5. In humans, RFC plays a This article contains supporting information online at https://www.pnas.org/lookup/suppl/ role in several diseases, such as cancer (11–13), Warsaw break- doi:10.1073/pnas.2007437117/-/DCSupplemental. age syndrome (14), cerebellar ataxia, neuropathy, and vestibular www.pnas.org/cgi/doi/10.1073/pnas.2007437117 PNAS Latest Articles | 1of10 Downloaded by guest on October 6, 2021 The pentameric clamp loader structure is broadly conserved. mutations perturb clamp loader function, as there is currently no The five subunits are named A through E going counterclockwise structure of the human RFC complex. around the assembly. Each of the five subunits consists of an Here we describe a cryogenic electron microscopy (cryo-EM) N-terminal AAA+ ATPase module, followed by an α-helical reconstruction of human RFC (hRFC) bound to PCNA. The “collar” domain that serves to oligomerize the complex (Fig. 1A). structure reveals that PCNA is closed, despite all active sites of The Rossman fold and Lid domains that comprise the AAA+ hRFC being bound to ATP analogs. The spiral of AAA+ module contain the catalytic residues for ATPase activity. Al- modules is constricted, which prevents opening of the clamp and though most of the catalytic machinery is used in cis, the B, C, blocks the DNA-binding region in the central chamber of the D, and E subunits all contain arginine finger residues that are clamp loader. We propose that this represents an autoinhibited provided in trans to complete the active site of a neighboring form of the clamp loader that occurs prior to clamp opening. subunit. Our work provides a framework for understanding the clamp ’ Structural studies have revealed critical intermediates for the loader s mechanism and function in human health. Escherichia clamp loading mechanism. Early structures of the Results coli clamp loader revealed the general organization of the complex (34, 35). A subsequent structure of a mutated form of Structure Determination of the hRFC:PCNA Complex. We sought to the Saccharomyces cerevisiae clamp loader RFC bound to PCNA obtain a structure of human RFC bound to PCNA by single- particle cryo-EM. We purified an hRFC construct with a trun- showed RFC in a collapsed and overtwisted spiral conformation cation of the A subunit’s N-terminal region (RFC1ΔN555, that is bound to a closed PCNA ring (36). This conformation was missing residues 1 to 555). The truncated version expresses in initially hypothesized to represent an intermediate toward the E. coli and results in higher yields of active protein than the full- end of the clamp loading reaction, with PCNA closed around length construct without sacrificing clamp loading activity (SI DNA and still bound to RFC prior to ATP hydrolysis. It has also Appendix, Fig. S1 A–C and ref. 40). This hRFC construct is been hypothesized that this conformation is an artifact of the similar to the Hutchinson–Gilford progeria syndrome variant, mutation of the arginine fingers that prevents proper assembly where the A subunit in hRFC is proteolytically truncated to a (31, 37). The structure of an off-pathway intermediate of the ∼75-kDa C-terminal fragment, removing the first ∼500 residues. E. coli – clamp loader bound to a primer template junction con- As expected (41, 42), our purified hRFC has highest ATPase “ ” firmed the notched-screwcap mode of DNA binding (38). Fi- activity in the presence of both the sliding clamp and primer– nally, the T4 phage clamp loader was crystallized with sliding template DNA (SI Appendix, Fig. S1D). For the rest of the pa- clamp and DNA, revealing how ATP hydrolysis is linked to per, we refer to this complex as hRFC. clamp closure (39). In order to visualize how the clamp loader interacts with the Despite many years of study, several central questions about sliding clamp, we formed a complex of hRFC with PCNA and the clamp loader mechanism remain unanswered.