Probing DNA clamps with single-molecule force spectroscopy

Wang, Lin; Xu, Xiaojun; Kumar, Ravindra; Maiti, Biswajit; Liu, C. Tony; Ivanov, Ivaylo; Lee, Tae Hee; Benkovic, Stephen J.

doi:10.1093/nar/gkt487

Cited by 10 publications

(12 citation statements)

References 38 publications

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“…Powerful new technologies are opening the door for much greater knowledge of clamp loader function, structure, and mechanism. Single‐molecule studies of clamp loader mechanism will be critical in the next era of investigation by accessing short‐lived and rare intermediates in the reaction. New developments in electron cryomicroscopy open the door for determining high‐resolution structures of new clamp loader complexes and hard‐to‐isolate reaction intermediates.…”

Section: Discussionmentioning

confidence: 99%

Review: The lord of the rings: Structure and mechanism of the sliding clamp loader

Kelch

2016

Biopolymers

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Sliding clamps are ring-shaped polymerase processivity factors that act as master regulators of cellular replication by coordinating multiple functions on DNA to ensure faithful transmission of genetic and epigenetic information. Dedicated AAA+ ATPase machines called clamp loaders actively place clamps on DNA, thereby governing clamp function by controlling when and where clamps are used. Clamp loaders are also important model systems for understanding the basic principles of AAA+ mechanism and function. After nearly 30 years of study, the ATP-dependent mechanism of opening and loading of clamps is now becoming clear. Here I review the structural and mechanistic aspects of the clamp loading process, as well as comment on questions that will be addressed by future studies. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 532-546, 2016.

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Section: Discussionmentioning

confidence: 99%

Review: The lord of the rings: Structure and mechanism of the sliding clamp loader

Kelch

2016

Biopolymers

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“…The θ subunit has no enzymatic function but binds and stabilizes ε 17 – 19 . The Pol III core (α, ε, and θ) binds to the DNA sliding clamp β 20 , 21 , essential for processive DNA synthesis. DNA synthesis by Pol III core – β complex is fast (600–1000 nucleotides per second), processive (>100,000 nucleotides per binding event) and at the same time highly precise (error rate ~1 per million) 16 , 22 – 24 .…”

Section: Introductionmentioning

confidence: 99%

Polymerization and editing modes of a high-fidelity DNA polymerase are linked by a well-defined path

et al. 2020

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Proofreading by replicative DNA polymerases is a fundamental mechanism ensuring DNA replication fidelity. In proofreading, mis-incorporated nucleotides are excised through the 3′-5′ exonuclease activity of the DNA polymerase holoenzyme. The exonuclease site is distal from the polymerization site, imposing stringent structural and kinetic requirements for efficient primer strand transfer. Yet, the molecular mechanism of this transfer is not known. Here we employ molecular simulations using recent cryo-EM structures and biochemical analyses to delineate an optimal free energy path connecting the polymerization and exonuclease states of E. coli replicative DNA polymerase Pol III. We identify structures for all intermediates, in which the transitioning primer strand is stabilized by conserved Pol III residues along the fingers, thumb and exonuclease domains. We demonstrate switching kinetics on a tens of milliseconds timescale and unveil a complete pol-to-exo switching mechanism, validated by targeted mutational experiments.

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“…coli Pol III β were used to show distinct opening mechanisms (unzipping in PCNA; abrupt cooperative disruption in E . coli Pol III β) [ 32 ]. Similarly, steered MD simulations suggest that human PCNA opens by unzipping of the dimer interface [ 33 ].…”

Section: Introductionmentioning

confidence: 99%

Dynamics of Open DNA Sliding Clamps

Oakley

2016

PLoS ONE

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A range of enzymes in DNA replication and repair bind to DNA-clamps: torus-shaped proteins that encircle double-stranded DNA and act as mobile tethers. Clamps from viruses (such as gp45 from the T4 bacteriophage) and eukaryotes (PCNAs) are homotrimers, each protomer containing two repeats of the DNA-clamp motif, while bacterial clamps (pol III β) are homodimers, each protomer containing three DNA-clamp motifs. Clamps need to be flexible enough to allow opening and loading onto primed DNA by clamp loader complexes. Equilibrium and steered molecular dynamics simulations have been used to study DNA-clamp conformation in open and closed forms. The E. coli and PCNA clamps appear to prefer closed, planar conformations. Remarkably, gp45 appears to prefer an open right-handed spiral conformation in solution, in agreement with previously reported biophysical data. The structural preferences of DNA clamps in solution have implications for understanding the duty cycle of clamp-loaders.

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Probing DNA clamps with single-molecule force spectroscopy

Cited by 10 publications

References 38 publications

Review: The lord of the rings: Structure and mechanism of the sliding clamp loader

Review: The lord of the rings: Structure and mechanism of the sliding clamp loader

Polymerization and editing modes of a high-fidelity DNA polymerase are linked by a well-defined path

Dynamics of Open DNA Sliding Clamps

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