The molecular coupling between substrate recognition and ATP turnover in a AAA+ hexameric helicase loader

Puri, Neha; Fernandez, Amy J.; Murray, Valerie L. O’Shea; McMillan, Sarah; Keck, James L.

doi:10.7554/elife.64232

Cited by 10 publications

(5 citation statements)

References 44 publications

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“…Our observations are in agreement with previous studies that found that the arginine switch residues of RFC do not likely play a direct role in activating ATP hydrolysis, but are important for the synergistic activation by both PCNA- and DNA binding ( Liu et al, 2017 ). An alternative route, involving a different arginine residue interacting with the ATPase active site, has recently been proposed for DnaC and extended to RFC ( Puri et al, 2021 ). However, we again do not see structural evidence supporting this mechanism.…”

Section: Discussionmentioning

confidence: 99%

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

Gaubitz

Liu

Pajak

et al. 2022

eLife

106

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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.

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Section: Discussionmentioning

confidence: 99%

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

Gaubitz

Liu

Pajak

et al. 2022

eLife

106

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“…4e ). It has been proposed that Tyr170 could act as a switch to control IstB ATPase activity, playing a part analogous to the Arg-coupler present in the loading factors of DNA polymerase clamps and the eukaryotic and prokaryotic replicative helicases 41 . Consistent with this proposal, a Y170A mutant maintained an integration activity similar to that of the wild-type protein (Fig.…”

Section: Ista β-Barrel Stimulates Atp Hydrolysismentioning

confidence: 99%

Molecular basis for transposase activation by a dedicated AAA+ ATPase

de la Gándara,

Spínola-Amilibia,

Araújo-Bazán

et al. 2024

Nature

Self Cite

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Transposases drive chromosomal rearrangements and the dissemination of drug-resistance genes and toxins1–3. Although some transposases act alone, many rely on dedicated AAA+ ATPase subunits that regulate site selectivity and catalytic function through poorly understood mechanisms. Using IS21 as a model transposase system, we show how an ATPase regulator uses nucleotide-controlled assembly and DNA deformation to enable structure-based site selectivity, transposase recruitment, and activation and integration. Solution and cryogenic electron microscopy studies show that the IstB ATPase self-assembles into an autoinhibited pentamer of dimers that tightly curves target DNA into a half-coil. Two of these decamers dimerize, which stabilizes the target nucleic acid into a kinked S-shaped configuration that engages the IstA transposase at the interface between the two IstB oligomers to form an approximately 1 MDa transpososome complex. Specific interactions stimulate regulator ATPase activity and trigger a large conformational change on the transposase that positions the catalytic site to perform DNA strand transfer. These studies help explain how AAA+ ATPase regulators—which are used by classical transposition systems such as Tn7, Mu and CRISPR-associated elements—can remodel their substrate DNA and cognate transposases to promote function.

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“…This distortion accumulates when more DnaC units are bound and eventually results in the helicase opening (Chodavarapu et al 2016 , Chase et al 2018 , Arias-Palomo et al 2019 , Nagata et al 2020 ). ATP binding in the DnaC ATPase domain stabilizes DnaB open conformation and is important for helicase activation (Arias-Palomo et al 2019 , Puri et al 2021 ). During loading, DnaB assumes a dilated conformation with a wide central channel, but it can also dynamically switch between dilated and constricted conformations as it travels on the ssDNA (Strycharska et al 2013 ).…”

Section: Dna Replication By the Replisomementioning

confidence: 99%

“…Encircling both DNA strands requires DnaB to be in a nonconstricted state when it is known to unwind more slowly (Strycharska et al 2013 ). Additionally, DnaC was shown to facilitate helicase unloading in an ATP-dependent manner (Puri et al 2021 ). It is, therefore, possible that slowing down the helicase somehow allows it to bind DnaC, which aids its detachment from DNA.…”

Section: Dna Replication By the Replisomementioning

confidence: 99%

Escherichia coli DNA replication: the old model organism still holds many surprises

Łazowski,

Woodgate,

Fijalkowska

2024

FEMS Microbiology Reviews

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Research on Escherichia coli DNA replication paved the groundwork for many breakthrough discoveries with important implications for our understanding of human molecular biology, due to the high level of conservation of key molecular processes involved. To this day, it attracts a lot of attention, partially by virtue of being an important model organism, but also because the understanding of factors influencing replication fidelity might be important for studies on the emergence of antibiotic resistance. Importantly, the wide access to high-resolution single-molecule and live-cell imaging, whole genome sequencing, and Cryo-EM techniques, which were greatly popularized in the last decade, allows us to revisit certain assumptions about the replisomes and offers very detailed insight into how they work. For many parts of the replisome, step-by-step mechanisms have been reconstituted, and some new players identified. This review summarizes the latest developments in the area, focusing on (a) the structure of the replisome and mechanisms of action of its components, (b) organization of replisome transactions and repair, (c) replisome dynamics, and (d) factors influencing the base and sugar fidelity of DNA synthesis.

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The molecular coupling between substrate recognition and ATP turnover in a AAA+ hexameric helicase loader

Cited by 10 publications

References 44 publications

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

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

Molecular basis for transposase activation by a dedicated AAA+ ATPase

Escherichia coli DNA replication: the old model organism still holds many surprises

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