The β Sliding Clamp Closes around DNA prior to Release by the Escherichia coli Clamp Loader γ Complex

Hayner, Jaclyn N.; Bloom, Linda B.

doi:10.1074/jbc.m112.406231

Cited by 29 publications

(35 citation statements)

References 49 publications

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“…Given that PCNA is closing is faster than PCNA release, the steps that govern the rates of these two reactions differ. This kinetic order of events is conserved in bacteria where the E. coli clamp closes around DNA faster than it is released by the clamp loader [53]. Interestingly, the rate of PCNA-MDCC release onto DNA as the result of clamp loading is about five times slower than previously reported for PCNA loading/release [23].…”

Section: Discussionmentioning

confidence: 92%

Kinetic analysis of PCNA clamp binding and release in the clamp loading reaction catalyzed by Saccharomyces cerevisiae replication factor C

Marzahn

Hayner

Meyer

et al. 2015

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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DNA polymerases require a sliding clamp to achieve processive DNA synthesis. The toroidal clamps are loaded onto DNA by clamp loaders, members of the AAA+ family of ATPases. These enzymes utilize the energy of ATP binding and hydrolysis to perform a variety of cellular functions. In this study, a clamp loader-clamp binding assay was developed to measure the rates of ATP-dependent clamp binding and ATP-hydrolysis-dependent clamp release for the S. cerevisiae clamp loader (RFC) and clamp (PCNA). Pre-steady-state kinetics of PCNA binding showed that although ATP binding to RFC increases affinity for PCNA, ATP binding rates and ATP-dependent conformational changes in RFC are fast relative to PCNA binding rates. Interestingly, RFC binds PCNA faster than the Escherichia coli γ complex clamp loader binds the β-clamp. In the process of loading clamps on DNA, RFC maintains contact with PCNA while PCNA closes, as the observed rate of PCNA closing is faster than the rate of PCNA release, precluding the possibility of an open clamp dissociating from DNA. Rates of clamp closing and release are not dependent on the rate of the DNA binding step and are also slower than reported rates of ATP hydrolysis, showing that these rates reflect unique intramolecular reaction steps in the clamp loading pathway.

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Section: Discussionmentioning

confidence: 92%

Kinetic analysis of PCNA clamp binding and release in the clamp loading reaction catalyzed by Saccharomyces cerevisiae replication factor C

Marzahn

Hayner

Meyer

et al. 2015

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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“…From crystal structures of the T4 phage clamp loader, a single nucleotide hydrolysis event in the B subunit results in clamp closure, although the clamp loader stays bound to both the clamp and DNA 56 (Figure 2, intermediate vii; Figure 4B). Moreover, biochemical data for the T4, E. coli and yeast clamp loaders indicate that clamp closure precedes clamp loader ejection 86,99,126,127 and that the first ATP hydrolysis event closes the clamp, 70 implying that this phenomenon is a general feature of all clamp loaders. Closure before ejection of the loader ensures that the clamp productively binds to DNA and avoids futile cycles of abortive loading.…”

Section: Atp Hydrolysis and Clamp Loader Ejectionmentioning

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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“…During clamp loading, a long-lived clamp loader-clamp complex will have more opportunity to survive failed attempts and, hence, enhance the overall efficiency of clamp loading onto the DNA strand. Consequently, ATP hydrolysis may be ultimately associated with dissociation of the clamp loader in addition to closure of the clamp (39,40). …”

Section: Resultsmentioning

confidence: 99%

“…Both the higher stability of the closed ring conformation and possible need for an active clamp loader participation in the ring opening are supported by the higher force and energy cost found here for disruption of the subunit interfacial interactions in β-clamp. Thus, despite the fact that S. cerevisiae PCNA clamp and E. coli β -clamp share many similar mechanistic aspects (40) in the clamp loading processes, there are many important distinctions between the two systems, such as the force and its associated detailed mechanisms of ring opening, which would elude the more common ensemble-averaged mechanistic studies.…”

Section: Discussionmentioning

confidence: 99%

Probing DNA clamps with single-molecule force spectroscopy

Wang

Kumar

et al. 2013

Nucleic Acids Research

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Detailed mechanisms of DNA clamps in prokaryotic and eukaryotic systems were investigated by probing their mechanics with single-molecule force spectroscopy. Specifically, the mechanical forces required for the Escherichia coli and Saccharomyces cerevisiae clamp opening were measured at the single-molecule level by optical tweezers. Steered molecular dynamics simulations further examined the forces involved in DNA clamp opening from the perspective of the interface binding energies associated with the clamp opening processes. In combination with additional molecular dynamics simulations, we identified the contact networks between the clamp subunits that contribute significantly to the interface stability of the S.cerevisiae and E. coli clamps. These studies provide a vivid picture of the mechanics and energy landscape of clamp opening and reveal how the prokaryotic and eukaryotic clamps function through different mechanisms.

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The β Sliding Clamp Closes around DNA prior to Release by the Escherichia coli Clamp Loader γ Complex

Cited by 29 publications

References 49 publications

Kinetic analysis of PCNA clamp binding and release in the clamp loading reaction catalyzed by Saccharomyces cerevisiae replication factor C

Kinetic analysis of PCNA clamp binding and release in the clamp loading reaction catalyzed by Saccharomyces cerevisiae replication factor C

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

Probing DNA clamps with single-molecule force spectroscopy

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