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Mike O'donnell - One of the best experts on this subject based on the ideXlab platform.
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The ATP sites of AAA+ Clamp loaders work together as a switch to assemble Clamps on DNA.
The Journal of biological chemistry, 2014Co-Authors: Melissa R. Marzahn, Jaclyn N. Hayner, Mike O'donnell, Jeff Finkelstein, Linda B BloomAbstract:Clamp loaders belong to a family of proteins known as ATPases associated with various cellular activities (AAA+). These proteins utilize the energy from ATP binding and hydrolysis to perform cellular functions. The Clamp loader is required to load the Clamp onto DNA for use by DNA polymerases to increase processivity. ATP binding and hydrolysis are coordinated by several key residues, including a conserved Lys located within the Walker A motif (or P-loop). This residue is required for each subunit to bind ATP. The specific function of each ATP molecule bound to the Saccharomyces cerevisiae Clamp loader is unknown. A series of point mutants, each lacking a single Walker A Lys residue, was generated to study the effects of abolishing ATP binding in individual Clamp loader subunits. A variety of biochemical assays were used to analyze the function of ATP binding during discrete steps of the Clamp loading reaction. All mutants reduced Clamp binding/opening to different degrees. Decreased Clamp binding activity was generally correlated with decreases in the population of open Clamps, suggesting that differences in the binding affinities of Walker A mutants stem from differences in stabilization of proliferating cell nuclear antigen in an open conformation. Walker A mutations had a smaller effect on DNA binding than Clamp binding/opening. Our data do not support a model in which each ATP site functions independently to regulate a different step in the Clamp loading cycle to coordinate these steps. Instead, the ATP sites work in unison to promote conformational changes in the Clamp loader that drive Clamp loading.
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Processivity Clamps in DNA Replication
Encyclopedia of Biological Chemistry, 2013Co-Authors: N.y. Yao, Mike O'donnellAbstract:Cells from all domains of life contain ring-shaped sliding Clamp proteins that encircle DNA and slide along it. Sliding Clamps were initially discovered by their ability to hold the chromosomal replicase to DNA for high processivity during replication of long chromosomes. Sliding Clamps are opened and closed around DNA by a Clamp-loading machine driven by ATP hydrolysis. Sliding Clamps also bind many other proteins including ligase, mismatch repair factors, certain nucleases, and numerous DNA polymerases involved in DNA repair. We briefly review the structure and function of sliding Clamps and Clamp-loading machines. We then describe how Clamps are utilized by the replicating apparatus to circumvent various obstacles encountered during replication.
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Replication factor C is a more effective proliferating cell nuclear antigen (PCNA) opener than the checkpoint Clamp loader, Rad24-RFC
Journal of Biological Chemistry, 2012Co-Authors: Jennifer A. Thompson, Mike O'donnell, Melissa R. Marzahn, Linda B BloomAbstract:Clamp loaders from all domains of life load Clamps onto DNA. The Clamp tethers DNA polymerases to DNA to increase the processivity of synthesis as well as the efficiency of replication. Here, we investigated proliferating cell nuclear antigen (PCNA) binding and opening by the Saccharomyces cerevisiae Clamp loader, replication factor C (RFC), and the DNA damage checkpoint Clamp loader, Rad24-RFC, using two separate fluorescence intensity-based assays. Analysis of PCNA opening by RFC revealed a two-step reaction in which RFC binds PCNA before opening PCNA rather than capturing Clamps that have transiently and spontaneously opened in solution. The affinity of RFC for PCNA is about an order of magnitude lower in the absence of ATP than in its presence. The affinity of Rad24-RFC for PCNA in the presence of ATP is about an order magnitude weaker than that of RFC for PCNA, similar to the RFC-PCNA interaction in the absence of ATP. Importantly, fewer open Clamp loader-Clamp complexes are formed when PCNA is bound by Rad24-RFC than when bound by RFC.
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How a DNA polymerase Clamp loader opens a sliding Clamp
Science, 2011Co-Authors: Brian A. Kelch, Debora L. Makino, Mike O'donnell, John KuriyanAbstract:Processive chromosomal replication relies on sliding DNA Clamps, which are loaded onto DNA by pentameric Clamp loader complexes belonging to the AAA+ family of adenosine triphosphatases (ATPases). We present structures for the ATP-bound state of the Clamp loader complex from bacteriophage T4, bound to an open Clamp and primer-template DNA. The Clamp loader traps a spiral conformation of the open Clamp so that both the loader and the Clamp match the helical symmetry of DNA. One structure reveals that ATP has been hydrolyzed in one subunit and suggests that Clamp closure and ejection of the loader involves disruption of the ATP-dependent match in symmetry. The structures explain how synergy among the loader, the Clamp, and DNA can trigger ATP hydrolysis and release of the closed Clamp on DNA.
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The Escherichia coli Clamp loader can actively pry open the β-sliding Clamp
Journal of Biological Chemistry, 2011Co-Authors: Christopher O. Paschall, Jaclyn N. Hayner, Ankita Chiraniya, Jennifer A. Thompson, Mike O'donnell, Arthur H Robbins, Melissa R. Marzahn, Robert Mckenna, Linda B BloomAbstract:Clamp loaders load ring-shaped sliding Clamps onto DNA. Once loaded onto DNA, sliding Clamps bind to DNA polymerases to increase the processivity of DNA synthesis. To load Clamps onto DNA, an open Clamp loader-Clamp complex must form. An unresolved question is whether Clamp loaders capture Clamps that have transiently opened or whether Clamp loaders bind closed Clamps and actively open Clamps. A simple fluorescence-based Clamp opening assay was developed to address this question and to determine how ATP binding contributes to Clamp opening. A direct comparison of real time binding and opening reactions revealed that the Escherichia coli γ complex binds β first and then opens the Clamp. Mutation of conserved "arginine fingers" in the γ complex that interact with bound ATP decreased Clamp opening activity showing that arginine fingers make an important contribution to the ATP-induced conformational changes that allow the Clamp loader to pry open the Clamp.
Linda B Bloom - One of the best experts on this subject based on the ideXlab platform.
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Mechanism of opening a sliding Clamp
Nucleic acids research, 2017Co-Authors: Lauren G. Douma, Jennifer K. England, Marcia Levitus, Linda B BloomAbstract:Clamp loaders load ring-shaped sliding Clamps onto DNA where the Clamps serve as processivity factors for DNA polymerases. In the first stage of Clamp loading, Clamp loaders bind and stabilize Clamps in an open conformation, and in the second stage, Clamp loaders place the open Clamps around DNA so that the Clamps encircle DNA. Here, the mechanism of the initial Clamp opening stage is investigated. Mutations were introduced into the Escherichia coli β-sliding Clamp that destabilize the dimer interface to determine whether the formation of an open Clamp loader–Clamp complex is dependent on spontaneous Clamp opening events. In other work, we showed that mutation of a positively charged Arg residue at the β-dimer interface and high NaCl concentrations destabilize the Clamp, but neither facilitates the formation of an open Clamp loader–Clamp complex in experiments presented here. Clamp opening reactions could be fit to a minimal three-step ‘bind-open-lock’ model in which the Clamp loader binds a closed Clamp, the Clamp opens, and subsequent conformational rearrangements ‘lock’ the Clamp loader–Clamp complex in a stable open conformation. Our results support a model in which the E. coli Clamp loader actively opens the β-sliding Clamp.
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Solution structure of an "open" E. coli Pol III Clamp loader sliding Clamp complex.
Journal of structural biology, 2016Co-Authors: Farzaneh Tondnevis, Linda B Bloom, Thomas M. Weiss, Tsutomu Matsui, Robert MckennaAbstract:Sliding Clamps are opened and loaded onto primer template junctions by Clamp loaders, and once loaded on DNA, confer processivity to replicative polymerases. Previously determined crystal structures of eukaryotic and T4 Clamp loader-Clamp complexes have captured the sliding Clamps in either closed or only partially open interface conformations. In these solution structure studies, we have captured for the first time the Clamp loader-sliding Clamp complex from Escherichia coli using size exclusion chromatography coupled to small angle X-ray scattering (SEC-SAXS). The data suggests the sliding Clamp is in an open conformation which is wide enough to permit duplex DNA binding. The data also provides information about spatial arrangement of the sliding Clamp with respect to the Clamp loader subunits and is compared to complex crystal structures determined from other organisms.
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kinetic analysis of pcna Clamp binding and release in the Clamp loading reaction catalyzed by saccharomyces cerevisiae replication factor c
Biochimica et Biophysica Acta, 2015Co-Authors: Melissa R. Marzahn, Jaclyn N. Hayner, Jennifer A Meyer, Linda B BloomAbstract:Abstract 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 Saccharomyces 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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The interplay of primer-template DNA phosphorylation status and single-stranded DNA binding proteins in directing Clamp loaders to the appropriate polarity of DNA
Nucleic Acids Research, 2014Co-Authors: Jaclyn N. Hayner, Lauren G. Douma, Linda B BloomAbstract:Sliding Clamps are loaded onto DNA by Clamp loaders to serve the critical role of coordinating various enzymes on DNA. Clamp loaders must quickly and efficiently load Clamps at primer/template (p/t) junctions containing a duplex region with a free 3′OH (3′DNA), but it is unclear how Clamp loaders target these sites. To measure the Escherichia coli and Saccharomyces cerevisiae Clamp loader specificity toward 3′DNA, fluorescent β and PCNA Clamps were used to measure Clamp closing triggered by DNA substrates of differing polarity, testing the role of both the 5′phosphate (5′P) and the presence of single-stranded binding proteins (SSBs). SSBs inhibit Clamp loading by both Clamp loaders on the incorrect polarity of DNA (5′DNA). The 5′P groups contribute selectivity to differing degrees for the two Clamp loaders, suggesting variations in the mechanism by which Clamp loaders target 3′DNA. Interestingly, the χ subunit of the E. coli Clamp loader is not required for SSB to inhibit Clamp loading on phosphorylated 5′DNA, showing that χ·SSB interactions are dispensable. These studies highlight a common role for SSBs in directing Clamp loaders to 3′DNA, as well as uncover nuances in the mechanisms by which SSBs perform this vital role.
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The ATP sites of AAA+ Clamp loaders work together as a switch to assemble Clamps on DNA.
The Journal of biological chemistry, 2014Co-Authors: Melissa R. Marzahn, Jaclyn N. Hayner, Mike O'donnell, Jeff Finkelstein, Linda B BloomAbstract:Clamp loaders belong to a family of proteins known as ATPases associated with various cellular activities (AAA+). These proteins utilize the energy from ATP binding and hydrolysis to perform cellular functions. The Clamp loader is required to load the Clamp onto DNA for use by DNA polymerases to increase processivity. ATP binding and hydrolysis are coordinated by several key residues, including a conserved Lys located within the Walker A motif (or P-loop). This residue is required for each subunit to bind ATP. The specific function of each ATP molecule bound to the Saccharomyces cerevisiae Clamp loader is unknown. A series of point mutants, each lacking a single Walker A Lys residue, was generated to study the effects of abolishing ATP binding in individual Clamp loader subunits. A variety of biochemical assays were used to analyze the function of ATP binding during discrete steps of the Clamp loading reaction. All mutants reduced Clamp binding/opening to different degrees. Decreased Clamp binding activity was generally correlated with decreases in the population of open Clamps, suggesting that differences in the binding affinities of Walker A mutants stem from differences in stabilization of proliferating cell nuclear antigen in an open conformation. Walker A mutations had a smaller effect on DNA binding than Clamp binding/opening. Our data do not support a model in which each ATP site functions independently to regulate a different step in the Clamp loading cycle to coordinate these steps. Instead, the ATP sites work in unison to promote conformational changes in the Clamp loader that drive Clamp loading.
John Kuriyan - One of the best experts on this subject based on the ideXlab platform.
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How a DNA polymerase Clamp loader opens a sliding Clamp
Science, 2011Co-Authors: Brian A. Kelch, Debora L. Makino, Mike O'donnell, John KuriyanAbstract:Processive chromosomal replication relies on sliding DNA Clamps, which are loaded onto DNA by pentameric Clamp loader complexes belonging to the AAA+ family of adenosine triphosphatases (ATPases). We present structures for the ATP-bound state of the Clamp loader complex from bacteriophage T4, bound to an open Clamp and primer-template DNA. The Clamp loader traps a spiral conformation of the open Clamp so that both the loader and the Clamp match the helical symmetry of DNA. One structure reveals that ATP has been hydrolyzed in one subunit and suggests that Clamp closure and ejection of the loader involves disruption of the ATP-dependent match in symmetry. The structures explain how synergy among the loader, the Clamp, and DNA can trigger ATP hydrolysis and release of the closed Clamp on DNA.
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mechanism of proliferating cell nuclear antigen Clamp opening by replication factor c
Journal of Biological Chemistry, 2006Co-Authors: Aaron M Johnson, John Kuriyan, Gregory D. Bowman, Mike OdonnellAbstract:Abstract The eukaryotic replication factor C (RFC) Clamp loader is an AAA+ spiral-shaped heteropentamer that opens and closes the circular proliferating cell nuclear antigen (PCNA) Clamp processivity factor on DNA. In this study, we examined the roles of individual RFC subunits in opening the PCNA Clamp. Interestingly, Rfc1, which occupies the position analogous to the δ Clamp-opening subunit in the Escherichia coli Clamp loader, is not required to open PCNA. The Rfc5 subunit is required to open PCNA. Consistent with this result, Rfc2·3·4·5 and Rfc2·5 subassemblies are capable of opening and unloading PCNA from circular DNA. Rfc5 is positioned opposite the PCNA interface from Rfc1, and therefore, its action with Rfc2 in opening PCNA indicates that PCNA is opened from the opposite side of the interface that the E. coli δ wrench acts upon. This marks a significant departure in the mechanism of eukaryotic and prokaryotic Clamp loaders. Interestingly, the Rad·RFC DNA damage checkpoint Clamp loader unloads PCNA Clamps from DNA. We propose that Rad·RFC may clear PCNA from DNA to facilitate shutdown of replication in the face of DNA damage.
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DNA polymerase Clamp loaders and DNA recognition
FEBS Letters, 2005Co-Authors: Gregory D. Bowman, Mike O'donnell, Eric R. Goedken, Steven L. Kazmirski, John KuriyanAbstract:Clamp loaders are heteropentameric ATPase assemblies that load sliding Clamps onto DNA and are critical for processive DNA replication. The DNA targets for Clamp loading are double-stranded/single-stranded junctions with recessed 3′ ends (primer-template junctions). Here, we briefly review the crystal structures of Clamp loader complexes and the insights they have provided into the mechanism of the Clamp loading process.
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Structural analysis of a eukaryotic sliding DNA Clamp–Clamp loader complex
Nature, 2004Co-Authors: Gregory D. Bowman, Mike O'donnell, John KuriyanAbstract:Sliding Clamps are ring-shaped proteins that encircle DNA and confer high processivity on DNA polymerases. Here we report the crystal structure of the five-protein Clamp loader complex (replication factor-C, RFC) of the yeast Saccharomyces cerevisiae, bound to the sliding Clamp (proliferating cell nuclear antigen, PCNA). Tight interfacial coordination of the ATP analogue ATP-gammaS by RFC results in a spiral arrangement of the ATPase domains of the Clamp loader above the PCNA ring. Placement of a model for primed DNA within the central hole of PCNA reveals a striking correspondence between the RFC spiral and the grooves of the DNA double helix. This model, in which the Clamp loader complex locks onto primed DNA in a screw-cap-like arrangement, provides a simple explanation for the process by which the engagement of primer-template junctions by the RFC:PCNA complex results in ATP hydrolysis and release of the sliding Clamp on DNA.
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Structural analysis of a eukaryotic sliding DNA Clamp-Clamp loader complex
Nature, 2004Co-Authors: Gregory D. Bowman, Mike O'donnell, John KuriyanAbstract:Sliding Clamps are ring-shaped proteins that encircle DNA and confer high processivity on DNA polymerases. Here we report the crystal structure of the five-protein Clamp loader complex (replication factor-C, RFC) of the yeast Saccharomyces cerevisiae, bound to the sliding Clamp (proliferating cell nuclear antigen, PCNA). Tight interfacial coordination of the ATP analogue ATP-gammaS by RFC results in a spiral arrangement of the ATPase domains of the Clamp loader above the PCNA ring. Placement of a model for primed DNA within the central hole of PCNA reveals a striking correspondence between the RFC spiral and the grooves of the DNA double helix. This model, in which the Clamp loader complex locks onto primed DNA in a screw-cap-like arrangement, provides a simple explanation for the process by which the engagement of primer-template junctions by the RFC:PCNA complex results in ATP hydrolysis and release of the sliding Clamp on DNA.
Gregory D. Bowman - One of the best experts on this subject based on the ideXlab platform.
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mechanism of proliferating cell nuclear antigen Clamp opening by replication factor c
Journal of Biological Chemistry, 2006Co-Authors: Aaron M Johnson, John Kuriyan, Gregory D. Bowman, Mike OdonnellAbstract:Abstract The eukaryotic replication factor C (RFC) Clamp loader is an AAA+ spiral-shaped heteropentamer that opens and closes the circular proliferating cell nuclear antigen (PCNA) Clamp processivity factor on DNA. In this study, we examined the roles of individual RFC subunits in opening the PCNA Clamp. Interestingly, Rfc1, which occupies the position analogous to the δ Clamp-opening subunit in the Escherichia coli Clamp loader, is not required to open PCNA. The Rfc5 subunit is required to open PCNA. Consistent with this result, Rfc2·3·4·5 and Rfc2·5 subassemblies are capable of opening and unloading PCNA from circular DNA. Rfc5 is positioned opposite the PCNA interface from Rfc1, and therefore, its action with Rfc2 in opening PCNA indicates that PCNA is opened from the opposite side of the interface that the E. coli δ wrench acts upon. This marks a significant departure in the mechanism of eukaryotic and prokaryotic Clamp loaders. Interestingly, the Rad·RFC DNA damage checkpoint Clamp loader unloads PCNA Clamps from DNA. We propose that Rad·RFC may clear PCNA from DNA to facilitate shutdown of replication in the face of DNA damage.
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DNA polymerase Clamp loaders and DNA recognition
FEBS Letters, 2005Co-Authors: Gregory D. Bowman, Mike O'donnell, Eric R. Goedken, Steven L. Kazmirski, John KuriyanAbstract:Clamp loaders are heteropentameric ATPase assemblies that load sliding Clamps onto DNA and are critical for processive DNA replication. The DNA targets for Clamp loading are double-stranded/single-stranded junctions with recessed 3′ ends (primer-template junctions). Here, we briefly review the crystal structures of Clamp loader complexes and the insights they have provided into the mechanism of the Clamp loading process.
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Structural analysis of a eukaryotic sliding DNA Clamp–Clamp loader complex
Nature, 2004Co-Authors: Gregory D. Bowman, Mike O'donnell, John KuriyanAbstract:Sliding Clamps are ring-shaped proteins that encircle DNA and confer high processivity on DNA polymerases. Here we report the crystal structure of the five-protein Clamp loader complex (replication factor-C, RFC) of the yeast Saccharomyces cerevisiae, bound to the sliding Clamp (proliferating cell nuclear antigen, PCNA). Tight interfacial coordination of the ATP analogue ATP-gammaS by RFC results in a spiral arrangement of the ATPase domains of the Clamp loader above the PCNA ring. Placement of a model for primed DNA within the central hole of PCNA reveals a striking correspondence between the RFC spiral and the grooves of the DNA double helix. This model, in which the Clamp loader complex locks onto primed DNA in a screw-cap-like arrangement, provides a simple explanation for the process by which the engagement of primer-template junctions by the RFC:PCNA complex results in ATP hydrolysis and release of the sliding Clamp on DNA.
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Structural analysis of a eukaryotic sliding DNA Clamp-Clamp loader complex
Nature, 2004Co-Authors: Gregory D. Bowman, Mike O'donnell, John KuriyanAbstract:Sliding Clamps are ring-shaped proteins that encircle DNA and confer high processivity on DNA polymerases. Here we report the crystal structure of the five-protein Clamp loader complex (replication factor-C, RFC) of the yeast Saccharomyces cerevisiae, bound to the sliding Clamp (proliferating cell nuclear antigen, PCNA). Tight interfacial coordination of the ATP analogue ATP-gammaS by RFC results in a spiral arrangement of the ATPase domains of the Clamp loader above the PCNA ring. Placement of a model for primed DNA within the central hole of PCNA reveals a striking correspondence between the RFC spiral and the grooves of the DNA double helix. This model, in which the Clamp loader complex locks onto primed DNA in a screw-cap-like arrangement, provides a simple explanation for the process by which the engagement of primer-template junctions by the RFC:PCNA complex results in ATP hydrolysis and release of the sliding Clamp on DNA.
Jaclyn N. Hayner - One of the best experts on this subject based on the ideXlab platform.
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kinetic analysis of pcna Clamp binding and release in the Clamp loading reaction catalyzed by saccharomyces cerevisiae replication factor c
Biochimica et Biophysica Acta, 2015Co-Authors: Melissa R. Marzahn, Jaclyn N. Hayner, Jennifer A Meyer, Linda B BloomAbstract:Abstract 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 Saccharomyces 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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The interplay of primer-template DNA phosphorylation status and single-stranded DNA binding proteins in directing Clamp loaders to the appropriate polarity of DNA
Nucleic Acids Research, 2014Co-Authors: Jaclyn N. Hayner, Lauren G. Douma, Linda B BloomAbstract:Sliding Clamps are loaded onto DNA by Clamp loaders to serve the critical role of coordinating various enzymes on DNA. Clamp loaders must quickly and efficiently load Clamps at primer/template (p/t) junctions containing a duplex region with a free 3′OH (3′DNA), but it is unclear how Clamp loaders target these sites. To measure the Escherichia coli and Saccharomyces cerevisiae Clamp loader specificity toward 3′DNA, fluorescent β and PCNA Clamps were used to measure Clamp closing triggered by DNA substrates of differing polarity, testing the role of both the 5′phosphate (5′P) and the presence of single-stranded binding proteins (SSBs). SSBs inhibit Clamp loading by both Clamp loaders on the incorrect polarity of DNA (5′DNA). The 5′P groups contribute selectivity to differing degrees for the two Clamp loaders, suggesting variations in the mechanism by which Clamp loaders target 3′DNA. Interestingly, the χ subunit of the E. coli Clamp loader is not required for SSB to inhibit Clamp loading on phosphorylated 5′DNA, showing that χ·SSB interactions are dispensable. These studies highlight a common role for SSBs in directing Clamp loaders to 3′DNA, as well as uncover nuances in the mechanisms by which SSBs perform this vital role.
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The ATP sites of AAA+ Clamp loaders work together as a switch to assemble Clamps on DNA.
The Journal of biological chemistry, 2014Co-Authors: Melissa R. Marzahn, Jaclyn N. Hayner, Mike O'donnell, Jeff Finkelstein, Linda B BloomAbstract:Clamp loaders belong to a family of proteins known as ATPases associated with various cellular activities (AAA+). These proteins utilize the energy from ATP binding and hydrolysis to perform cellular functions. The Clamp loader is required to load the Clamp onto DNA for use by DNA polymerases to increase processivity. ATP binding and hydrolysis are coordinated by several key residues, including a conserved Lys located within the Walker A motif (or P-loop). This residue is required for each subunit to bind ATP. The specific function of each ATP molecule bound to the Saccharomyces cerevisiae Clamp loader is unknown. A series of point mutants, each lacking a single Walker A Lys residue, was generated to study the effects of abolishing ATP binding in individual Clamp loader subunits. A variety of biochemical assays were used to analyze the function of ATP binding during discrete steps of the Clamp loading reaction. All mutants reduced Clamp binding/opening to different degrees. Decreased Clamp binding activity was generally correlated with decreases in the population of open Clamps, suggesting that differences in the binding affinities of Walker A mutants stem from differences in stabilization of proliferating cell nuclear antigen in an open conformation. Walker A mutations had a smaller effect on DNA binding than Clamp binding/opening. Our data do not support a model in which each ATP site functions independently to regulate a different step in the Clamp loading cycle to coordinate these steps. Instead, the ATP sites work in unison to promote conformational changes in the Clamp loader that drive Clamp loading.
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The Escherichia coli Clamp loader can actively pry open the β-sliding Clamp
Journal of Biological Chemistry, 2011Co-Authors: Christopher O. Paschall, Jaclyn N. Hayner, Ankita Chiraniya, Jennifer A. Thompson, Mike O'donnell, Arthur H Robbins, Melissa R. Marzahn, Robert Mckenna, Linda B BloomAbstract:Clamp loaders load ring-shaped sliding Clamps onto DNA. Once loaded onto DNA, sliding Clamps bind to DNA polymerases to increase the processivity of DNA synthesis. To load Clamps onto DNA, an open Clamp loader-Clamp complex must form. An unresolved question is whether Clamp loaders capture Clamps that have transiently opened or whether Clamp loaders bind closed Clamps and actively open Clamps. A simple fluorescence-based Clamp opening assay was developed to address this question and to determine how ATP binding contributes to Clamp opening. A direct comparison of real time binding and opening reactions revealed that the Escherichia coli γ complex binds β first and then opens the Clamp. Mutation of conserved "arginine fingers" in the γ complex that interact with bound ATP decreased Clamp opening activity showing that arginine fingers make an important contribution to the ATP-induced conformational changes that allow the Clamp loader to pry open the Clamp.
