Allosteric communication in DNA polymerase clamp loaders relies on a critical hydrogen-bonded junction

106

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 S. 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.

Section: Discussionmentioning

confidence: 99%

Cryo-EM structures reveal high-resolution mechanism of a DNA polymerase sliding clamp loader

Gaubitz

Liu

Pajak

et al. 2022

106

“…In support of this hypothesis, we observe that the arginine switch residue is flipped into the active conformation in most of our structures ( Figure 6—figure supplement 2 ). The DNA also rigidifies the AAA+ spiral, which is likely a key factor in ATPase activity ( Subramanian et al, 2021 ). Finally, ATP hydrolysis and P i release triggers clamp closure and ejection of the clamp loader, which can then recycle to load another clamp (step 7).…”

Section: Discussionmentioning

confidence: 99%

A second DNA binding site on RFC facilitates clamp loading at gapped or nicked DNA

Liu

Gaubitz

Pajak

et al. 2022

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 ATP sites of AAA + oligomers, many of which are used in DNA replication, are located at subunit interfaces ( Erzberger and Berger, 2006 ), including the bacterial and eukaryotic RFC clamp loaders ( Bowman et al, 2004 ; Jeruzalmi et al, 2001 ). Interfacial ATP sites makes possible the coordination among subunits during hydrolysis and, in fact, recent deep mutagenesis of a clamp loader pentamer reveals a communication network among subunits needed to achieve the clamp loading reaction ( Subramanian et al, 2021 ).…”

Section: Introductionmentioning

confidence: 99%

Cryo-EM structures reveal that RFC recognizes both the 3′- and 5′-DNA ends to load PCNA onto gaps for DNA repair

Zheng

Georgescu

Yao

et al. 2022

Self Cite

RFC uses ATP to assemble PCNA onto primed sites for replicative DNA polymerases δ and ε. The RFC pentamer forms a central chamber that binds 3′ ss/ds DNA junctions to load PCNA onto DNA during replication. We show here five structures that identify a second DNA binding site in RFC that binds a 5′ duplex. This 5′ DNA site is located between the N-terminal BRCT domain and AAA+ module of the large Rfc1 subunit. Our structures reveal ideal binding to a 7-nt gap, which includes 2 bp unwound by the clamp loader. Biochemical studies show enhanced binding to 5 and 10 nt gaps, consistent with the structural results. Because both 3′ and 5′ ends are present at a ssDNA gap, we propose that the 5′ site facilitates RFC’s PCNA loading activity at a DNA damage-induced gap to recruit gap-filling polymerases. These findings are consistent with genetic studies showing that base excision repair of gaps greater than 1 base requires PCNA and involves the 5′ DNA binding domain of Rfc1. We further observe that a 5′ end facilitates PCNA loading at an RPA coated 30-nt gap, suggesting a potential role of the RFC 5′-DNA site in lagging strand DNA synthesis.