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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, 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.
Mike Odonnell - One of the best experts on this subject based on the ideXlab platform.
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a novel function for the conserved glutamate residue in the walker b motif of replication factor c
Genes, 2013Co-Authors: Ankita Chiraniya, Mike Odonnell, Jeff Finkelstein, Linda B BloomAbstract:In all domains of life, sliding clamps tether DNA polymerases to DNA to increase the processivity of synthesis. Clamp Loaders load clamps onto DNA in a multi-step process that requires ATP binding and hydrolysis. Like other AAA+ proteins, clamp Loaders contain conserved Walker A and Walker B sequence motifs, which participate in ATP binding and hydrolysis, respectively. Mutation of the glutamate residue in Walker B motifs (or DExx-boxes) in AAA+ proteins typically reduces ATP hydrolysis by as much as a couple orders of magnitude, but has no effect on ATP binding. Here, the Walker B Glu in each of the four active ATP sites of the eukaryotic clamp loader, RFC, was mutated to Gln and Ala separately, and ATP binding- and hydrolysis-dependent activities of the quadruple mutant clamp Loaders were characterized. Fluorescence-based assays were used to measure individual reaction steps required for clamp loading including clamp binding, clamp opening, DNA binding and ATP hydrolysis. Our results show that the Walker B mutations affect ATP-binding-dependent interactions of RFC with the clamp and DNA in addition to reducing ligand-dependent ATP hydrolysis activity. Here, we show that the Walker B glutamate is required for ATP-dependent ligand binding activity, a previously unknown function for this conserved Glu residue in RFC.
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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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clamp Loaders and replication initiation
Current Opinion in Structural Biology, 2006Co-Authors: Mike Odonnell, John KuriyanAbstract:Clamp Loaders are ATP-driven multiprotein machines that couple ATP hydrolysis to the opening and closing of a circular protein ring around DNA. This ring-shaped clamp slides along DNA, and interacts with numerous proteins involved in DNA replication, DNA repair and cell cycle control. Recently determined structures of clamp loader complexes from prokaryotic and eukaryotic sources have revealed exciting new details of how these complex AAA+ machines perform this essential clamp loading function.
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cellular dna replicases components and dynamics at the replication fork
Annual Review of Biochemistry, 2005Co-Authors: Aaron M Johnson, Mike OdonnellAbstract:▪ Abstract Chromosomal DNA replicases are multicomponent machines that have evolved clever strategies to perform their function. Although the structure of DNA is elegant in its simplicity, the job of duplicating it is far from simple. At the heart of the replicase machinery is a heteropentameric AAA+ clamp-loading machine that couples ATP hydrolysis to load circular clamp proteins onto DNA. The clamps encircle DNA and hold polymerases to the template for processive action. Clamp-loader and sliding clamp structures have been solved in both prokaryotic and eukaryotic systems. The heteropentameric clamp Loaders are circular oligomers, reflecting the circular shape of their respective clamp substrates. Clamps and clamp Loaders also function in other DNA metabolic processes, including repair, checkpoint mechanisms, and cell cycle progression. Twin polymerases and clamps coordinate their actions with a clamp loader and yet other proteins to form a replisome machine that advances the replication fork.
John Kuriyan - 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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clamp Loaders and replication initiation
Current Opinion in Structural Biology, 2006Co-Authors: Mike Odonnell, John KuriyanAbstract:Clamp Loaders are ATP-driven multiprotein machines that couple ATP hydrolysis to the opening and closing of a circular protein ring around DNA. This ring-shaped clamp slides along DNA, and interacts with numerous proteins involved in DNA replication, DNA repair and cell cycle control. Recently determined structures of clamp loader complexes from prokaryotic and eukaryotic sources have revealed exciting new details of how these complex AAA+ machines perform this essential clamp loading function.
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DNA polymerase clamp Loaders and DNA recognition
FEBS Letters, 2005Co-Authors: Gregory D. Bowman, 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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Motors and switches: AAA+ machines within the replisome
Nature Reviews Molecular Cell Biology, 2002Co-Authors: Megan J. Davey, David Jeruzalmi, John KuriyanAbstract:The Escherichia coli clamp loader, γ-complex, loads the ring-shaped β-clamp onto DNA in an ATP driven reaction in which the clamp is cracked open, brought to a primed site and closed around the DNA. AAA+ (ATPases associated with a variety of cellular activities) proteins are involved in many different aspects of DNA metabolism. The γ- and δ′-subunits of the γ-complex are AAA+ proteins and so are clamp-loader subunits from eukaryotes, archaea and T4 bacteriophage. The recent crystal structure of the γ_3δδ′-assembly, an active clamp loader, indicates that the clamp loader is a pentameric ring. A striking feature of this assembly is the location of ATP sites at the interfaces of the subunits. Clamp Loaders from eukaryotes, archaea and T4 bacteriophage share similarities to the E. coli γ-complex and models similar to that of of the E. coli γ-complex can be made for each of these clamp Loaders. Many other replication proteins are AAA+ proteins, including the replication initiation proteins, DnaA, the origin recognition complex (ORC), Mcm2–7, Cdc6 and DnaC. The similarities between the γ-complex subunits and other AAA+ proteins that are involved in replication initiation, lead to a model for the function of the replication-initiation proteins based on γ-complex structural and biochemical data. Clamp Loaders are required to load the ring-shaped clamps that tether replicative DNA polymerases onto DNA. Recently solved crystal structures, along with a series of biochemical studies, have provided a detailed understanding of the clamp loading reaction. In particular, studies of the Escherichia coli clamp loader — an AAA+ machine — have provided insights into the architecture of clamp Loaders from eukaryotes, bacteriophage T4 and archaea. Other AAA+ proteins are also involved in the initiation of DNA replication, and studies of the E. coli clamp loader indicate mechanisms by which these proteins might function.
Melissa R. Marzahn - 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 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, 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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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, 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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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, 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.
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, 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, 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.