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

Subramanian, Subu; Gorday, Kent; Marcus, Kendra; Orellana, Matthew R; Ren, Peter; Luo, Xiao Ran; O'Donnell, Mike; Kuriyan, John

doi:10.1101/2020.12.31.424921

Cited by 7 publications

(23 citation statements)

References 89 publications

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“…In support of this hypothesis, we observe that the arginine switch residue is flipped into the active conformation in most of our structures (Supplemental Figure 6.2). 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.…”

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

Preprint

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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.

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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

Preprint

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“…The protein gp62 occupies the A position and has four identical gp44 subunits at positions B, C, D, and E. gp62 binds two clamp subunitsunlike gp44, which binds one (Kelch et al 2011). High throughput mutagenesis of the T4 bacteriophage clamp loader revealed that regions not involved in catalysis or binding could tolerate mutations (Subramanian et al 2021). The exception to this trend was a mutationally sensitive glutamine residue (Gln 118) that is spatially distant from both catalytic and interfacial sites.…”

Section: Clade 1: Clamp Loader Cladementioning

confidence: 99%

“…Hydrogen bonds formed by this residue appear to rigidly fasten two helices in the AAAþ core domain and connect DNA bound in the central channel of the clamp loader to the nucleotide at the catalytic site. This glutamine-mediated hydrogen bonding network is present in AAAþ proteins outside of Clade 1 and may thus be an important general feature of these proteins as it appears to link domains responsible for ATP binding and/or hydrolysis across neighboring protomers (Subramanian et al 2021).…”

Section: Clade 1: Clamp Loader Cladementioning

confidence: 99%

The AAA+ superfamily: a review of the structural and mechanistic principles of these molecular machines

Khan

White

Brunger

2021

Critical Reviews in Biochemistry and Molecular Biology

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ATPases associated with diverse cellular activities (AAAþ proteins) are a superfamily of proteins found throughout all domains of life. The hallmark of this family is a conserved AAAþ domain responsible for a diverse range of cellular activities. Typically, AAAþ proteins transduce chemical energy from the hydrolysis of ATP into mechanical energy through conformational change, which can drive a variety of biological processes. AAAþ proteins operate in a variety of cellular contexts with diverse functions including disassembly of SNARE proteins, protein quality control, DNA replication, ribosome assembly, and viral replication. This breadth of function illustrates both the importance of AAAþ proteins in health and disease and emphasizes the importance of understanding conserved mechanisms of chemo-mechanical energy transduction. This review is divided into three major portions. First, the core AAAþ fold is presented. Next, the seven different clades of AAAþ proteins and structural details and reclassification pertaining to proteins in each clade are described. Finally, two well-known AAAþ proteins, NSF and its close relative p97, are reviewed in detail.

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“…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

Zhang

Georgescue

Yao

et al. 2022

Preprint

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RFC uses ATP to assemble PCNA onto primed sites for replicative DNA polymerases delta and epsilon;. The RFC pentamer forms a central chamber that binds a 3 primed DNA junction to load PCNA onto DNA during replication. We show here five structures revealing a novel second DNA binding site in RFC that binds a 5 prime junction. This 5 prime DNA site is located in the N-terminal BRCT domain and AAA module of the large Rfc1 subunit. It appears that 5 prime DNA binds after 3 prime DNA binding to RFC. 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 nucleotide gaps, consistent with the structural results. We propose that the 5 prime DNA site facilitates RFC 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 prime DNA binding domain of Rfc1. We further observe that a 5 prime site facilitates PCNA loading at an RPA coated 50-nt gap, suggesting a potential role of the RFC 5 prime DNA site at longer gaps including during lagging strand DNA synthesis.

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

Cited by 7 publications

References 89 publications

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

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

The AAA+ superfamily: a review of the structural and mechanistic principles of these molecular machines

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

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