Clamp loaders place circular sliding clamp proteins onto DNA so that clamp-binding partner proteins can synthesize, scan, and repair the genome. DNA with nicks or small single-stranded gaps are common clamp-loading targets in DNA repair, yet these substrates would be sterically blocked given the known mechanism for binding of primer-template DNA. Here, we report the discovery of a second DNA binding site in the yeast clamp loader Replication Factor C (RFC) that aids in binding to nicked or gapped DNA. This DNA binding site is on the external surface and is only accessible in the open conformation of RFC. Initial DNA binding at this site thus provides access to the primary DNA binding site in the central chamber. Furthermore, we identify that this site can partially unwind DNA to create an extended single-stranded gap for DNA binding in RFC's central chamber and subsequent ATPase activation. Finally, we show that deletion of the BRCT domain, a major component of the external DNA binding site, results in defective yeast growth in the presence of DNA damage where nicked or gapped DNA intermediates occur. We propose that RFC’s external DNA binding site acts to enhance DNA binding and clamp loading, particularly at DNA architectures typically found in DNA repair.
The reported cryo-EM map and atomic coordinates have been deposited in the Electron Microscopy Data Bank (entry numbers EMD-26280EMD-26298EMD-26302EMD-26297) and the Protein Data Bank (ID codes 7U1A, 7U1P, 7U19).
RFC:PCNA bound to dsDNA with a ssDNA gap of six nucleotidesPDB 7U1A, EMD-26298.
RFC:PCNA bound to DNA with a ssDNA gap of five nucleotidesPDB 7U1P, EMD-26302.
RFC:PCNA bound to nicked DNAPDB 7U19, EMD-26297.
- Brian A Kelch
- Christl Gaubitz
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Bruce Stillman, Cold Spring Harbor Laboratory, United States
© 2022, Liu et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Sliding clamps are ring-shaped protein complexes that are integral to the DNA replication machinery of all life. Sliding clamps are opened and installed onto DNA by clamp loader AAA+ ATPase complexes. However, how a clamp loader opens and closes the sliding clamp around DNA is still unknown. Here, we describe structures of the Saccharomyces cerevisiae clamp loader Replication Factor C (RFC) bound to its cognate sliding clamp Proliferating Cell Nuclear Antigen (PCNA) en route to successful loading. RFC first binds to PCNA in a dynamic, closed conformation that blocks both ATPase activity and DNA binding. RFC then opens the PCNA ring through a large-scale ‘crab-claw’ expansion of both RFC and PCNA that explains how RFC prefers initial binding of PCNA over DNA. Next, the open RFC:PCNA complex binds DNA and interrogates the primer-template junction using a surprising base-flipping mechanism. Our structures indicate that initial PCNA opening and subsequent closure around DNA do not require ATP hydrolysis, but are driven by binding energy. ATP hydrolysis, which is necessary for RFC release, is triggered by interactions with both PCNA and DNA, explaining RFC’s switch-like ATPase activity. Our work reveals how a AAA+ machine undergoes dramatic conformational changes for achieving binding preference and substrate remodeling.
The Endosomal Sorting Complexes Required for Transport (ESCRT) machinery mediates the membrane fission step that completes cytokinetic abscission and separates dividing cells. Filaments composed of ESCRT-III subunits constrict membranes of the intercellular bridge midbody to the abscission point. These filaments also bind and recruit cofactors whose activities help execute abscission and/or delay abscission timing in response to mitotic errors via the NoCut/Abscission checkpoint. We previously showed that the ESCRT-III subunit IST1 binds the cysteine protease CAPN7 (Calpain-7) and that CAPN7 is required for both efficient abscission and NoCut checkpoint maintenance (Wenzel et al., 2022). Here, we report biochemical and crystallographic studies showing that the tandem MIT domains of CAPN7 bind simultaneously to two distinct IST1 MIT interaction motifs. Structure-guided point mutations in either CAPN7 MIT domain disrupted IST1 binding in vitro and in cells, and depletion/rescue experiments showed that the CAPN7-IST1 interaction is required for: 1) CAPN7 recruitment to midbodies, 2) efficient abscission, and 3) NoCut checkpoint arrest. CAPN7 proteolytic activity is also required for abscission and checkpoint maintenance. Hence, IST1 recruits CAPN7 to midbodies, where its proteolytic activity is required to regulate and complete abscission.