The Experts below are selected from a list of 17109 Experts worldwide ranked by ideXlab platform
Weylim Wong - One of the best experts on this subject based on the ideXlab platform.
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attachment ability of a clamp bearing fish parasite diplozoon paradoxum monogenea on gills of the common bream abramis brama
The Journal of Experimental Biology, 2013Co-Authors: Weylim WongAbstract:Monogeneans, which are mainly fish ectoparasites, use various types of haptoral (posterior) attachment apparatus to secure their attachment onto their hosts. However, it remains unclear how strongly a monogenean can attach onto its host. In the present study, we aimed for the first time to (1) measure pull-off forces required to detach a pair of clamp-bearing monogeneans, Diplozoon paradoxum from gills of Abramis brama and (2) determine the contribution of muscles to the clamp movements. An average force of 6.1 ± 2.7 mN (about 246 times of the animals’ weight) was required to dislodge a paired D. paradoxum vertically from the gills. There were significant differences (p < 0.05, Tukey test) between the widths of clamp openings in D. paradoxum treated in three different solutions: the widest clamp openings were observed in the monogeneans treated in 100 mM potassium chloride solution (58.26 ± 13.44 µm), followed by those treated in 20 mM magnesium chloride solution (37.91 ± 7.58 µm), and finally those treated in filtered lake water (20.16 ± 8.63 µm). This suggests that the closing of the clamps is probably not due to the continuous contraction of extrinsic muscles but caused by the elasticity of the clamp material and that muscle activity is required for clamp opening.
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attachment ability of a clamp bearing fish parasite diplozoon paradoxum monogenea on gills of the common bream abramis brama
The Journal of Experimental Biology, 2013Co-Authors: Weylim Wong, Stanislav N GorbAbstract:Monogeneans, which are mainly fish ectoparasites, use various types of haptoral (posterior) attachment apparatus to secure their attachment onto their hosts. However, it remains unclear how strongly a monogenean can attach onto its host. In the present study, we aimed for the first time to (1) measure pull-off forces required to detach a pair of clamp-bearing monogeneans, Diplozoon paradoxum, from gills of Abramis brama and (2) determine the contribution of muscles to the clamp movements. A mean force of 6.1±2.7 mN (~246 times the animals' weight) was required to dislodge a paired D. paradoxum vertically from the gills. There were significant differences (P<0.05, Tukey test) between the widths of clamp openings in D. paradoxum treated in three different solutions: the widest clamp openings were observed in the monogeneans treated in 100 mmol l(-1) potassium chloride solution (58.26±13.44 μm), followed by those treated in 20 mmol l(-1) magnesium chloride solution (37.91±7.58 μm), and finally those treated in filtered lake water (20.16±8.63 μm). This suggests that the closing of the clamps is probably not due to the continuous contraction of extrinsic muscles but is caused by the elasticity of the clamp material and that muscle activity is required for clamp opening.
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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crystal structure of a dna polymerase sliding clamp from a gram positive bacterium
BMC Structural Biology, 2006Co-Authors: Maria A Argiriadi, Mike Odonnell, John Kuriyan, Eric R. Goedken, Irina BruckAbstract:Background Sliding DNA clamps are processivity factors that are required for efficient DNA replication. DNA polymerases maintain proximity to nucleic acid templates by interacting with sliding clamps that encircle DNA and thereby link the polymerase enzyme to the DNA substrate. Although the structures of sliding clamps from Gram-negative bacteria (E. coli), eukaryotes, archaea, and T4-like bacteriophages are well-known, the structure of a sliding clamp from Gram-positive bacteria has not been reported previously.
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out of plane motions in open sliding clamps molecular dynamics simulations of eukaryotic and archaeal proliferating cell nuclear antigen
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Steven L. Kazmirski, Mike Odonnell, John Kuriyan, Gregory D. Bowman, Yanxiang ZhaoAbstract:Sliding clamps are ring-like multimeric proteins that encircle duplex DNA and serve as mobile DNA-bound platforms that are essential for efficient DNA replication and repair. Sliding clamps are placed on DNA by clamp loader complexes, in which the clamp-interacting elements are organized in a right-handed spiral assembly. To understand how the flat, ring-like clamps might interact with the spiral interaction surface of the clamp loader complex, we have performed molecular dynamics simulations of sliding clamps (proliferating cell nuclear antigen from the budding yeast, humans, and an archaeal species) in which we have removed one of the three subunits so as to release the constraint of ring closure. The simulations reveal significant structural fluctuations corresponding to lateral opening and out-of-plane distortions of the clamp, which result principally from bending and twisting of the β-sheets that span the intermolecular interfaces, with smaller but similar contributions from β-sheets that span the intramolecular interfaces within each subunit. With the integrity of these β-sheets intact, the predominant fluctuations seen in the simulations are oscillations between lateral openings and right-handed spirals. The tendency for clamps to adopt a right-handed spiral conformation implies that once opened, the conformation of the clamp can easily match the spiraling of clamp loader subunits, a feature that is intrinsic to the recognition of DNA and subsequent hydrolysis of ATP by the clamp-bound clamp loader complex.
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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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Structural analysis of a eukaryotic sliding DNA clamp-clamp loader complex
Nature, 2004Co-Authors: Gregory D. Bowman, 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.
Linda B Bloom - One of the best experts on this subject based on the ideXlab platform.
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potassium glutamate and glycine betaine induce self assembly of the pcna and β sliding clamps
Biophysical Journal, 2021Co-Authors: Anirban Purohit, Linda B Bloom, Lauren G. Douma, Marcia LevitusAbstract:Abstract Sliding clamps are oligomeric ring-shaped proteins that increase the efficiency of DNA replication. The stability of the Escherichia coli β-clamp, a homodimer, is particularly remarkable. The dissociation equilibrium constant of the β-clamp is of the order of 10 pM in buffers of moderate ionic strength. Coulombic electrostatic interactions have been shown to contribute to this remarkable stability. Increasing NaCl concentration in the assay buffer results in decreased dimer stability and faster subunit dissociation kinetics in a way consistent with simple charge-screening models. Here, we examine non-Coulombic ionic effects on the oligomerization properties of sliding clamps. We determined relative diffusion coefficients of two sliding clamps using fluorescence correlation spectroscopy. Replacing NaCl by KGlu, the primary cytoplasmic salt in E. coli, results in a decrease of the diffusion coefficient of these proteins consistent with the formation of protein assemblies. The UV-vis spectrum of the β-clamp labeled with tetramethylrhodamine shows the characteristic absorption band of dimers of rhodamine when KGlu is present in the buffer. This suggests that KGlu induces the formation of assemblies that involve two or more rings stacked face-to-face. Results can be quantitatively explained on the basis of unfavorable interactions between KGlu and the functional groups on the protein surface, which drive biomolecular processes that bury exposed surface. Similar results were obtained with the Saccharomyces cerevisiae PCNA sliding clamp, suggesting that KGlu effects are not specific to the β-clamp. Clamp association is also promoted by glycine betaine, a zwitterionic compound that accumulates intracellularly when E. coli is exposed to high concentrations of extracellular solute. Possible biological implications are discussed.
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potassium glutamate and glycine betaine induce self assembly of sliding clamps into higher order oligomers
bioRxiv, 2020Co-Authors: Anirban Purohit, Linda B Bloom, Lauren G. Douma, Marcia LevitusAbstract:Sliding clamps are oligomeric ring-shaped proteins that increase the efficiency of DNA replication. The stability of the Escherichia coli β-clamp, a homodimer, is particularly remarkable. The dissociation equilibrium constant of β is of the order of 10 pM in buffers of moderate ionic strength. Coulombic electrostatic interactions have been shown to contribute to this remarkable stability. Increasing NaCl concentration in the assay buffer results in decreased dimer stability and faster subunit dissociation kinetics in a way consistent with simple charge-screening models. Here, we examine non-Coulombic ionic effects on the oligomerization properties of sliding clamps. Replacing NaCl by KGlu, the primary cytoplasmic salt in E. coli, results in the formation of assemblies that involve two or more rings stacked face-to-face. Results can be quantitatively explained on the basis of unfavorable interactions between KGlu and the functional groups on the protein surface, which drive biomolecular processes that bury exposed surface. Similar results were obtained with the S. cerevisiae PCNA sliding clamp, suggesting that KGlu effects are not specific to β. Clamp association is also promoted by glycine betaine, a zwitterionic compound that accumulates intracellularly when E. coli is exposed to high concentrations of extracellular solute. Possible biological implications are discussed.
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electrostatic interactions at the dimer interface stabilize the e coli β sliding clamp
Biophysical Journal, 2017Co-Authors: Anirban Purohit, Linda B Bloom, Lauren G. Douma, Jennifer K England, Farzaneh Tondnevis, Marcia LevitusAbstract:Sliding clamps are ring-shaped oligomeric proteins that encircle DNA and associate with DNA polymerases for processive DNA replication. The dimeric Escherichia coli β-clamp is closed in solution but must adopt an open conformation to be assembled onto DNA by a clamp loader. To determine what factors contribute to the stability of the dimer interfaces in the closed conformation and how clamp dynamics contribute to formation of the open conformation, we identified conditions that destabilized the dimer and measured the effects of these conditions on clamp dynamics. We characterized the role of electrostatic interactions in stabilizing the β-clamp interface. Increasing salt concentration results in decreased dimer stability and faster subunit dissociation kinetics. The equilibrium dissociation constant of the dimeric clamp varies with salt concentration as predicted by simple charge-screening models, indicating that charged amino acids contribute to the remarkable stability of the interface at physiological salt concentrations. Mutation of a charged residue at the interface (Arg-103) weakens the interface significantly, whereas effects are negligible when a hydrophilic (Ser-109) or a hydrophobic (Ile-305) amino acid is mutated instead. It has been suggested that clamp opening by the clamp loader takes advantage of spontaneous opening-closing fluctuations at the clamp's interface, but our time-resolved fluorescence and fluorescence correlation experiments rule out conformational fluctuations that lead to a significant fraction of open states.
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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.
Stanislav N Gorb - One of the best experts on this subject based on the ideXlab platform.
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attachment ability of a clamp bearing fish parasite diplozoon paradoxum monogenea on gills of the common bream abramis brama
The Journal of Experimental Biology, 2013Co-Authors: Weylim Wong, Stanislav N GorbAbstract:Monogeneans, which are mainly fish ectoparasites, use various types of haptoral (posterior) attachment apparatus to secure their attachment onto their hosts. However, it remains unclear how strongly a monogenean can attach onto its host. In the present study, we aimed for the first time to (1) measure pull-off forces required to detach a pair of clamp-bearing monogeneans, Diplozoon paradoxum, from gills of Abramis brama and (2) determine the contribution of muscles to the clamp movements. A mean force of 6.1±2.7 mN (~246 times the animals' weight) was required to dislodge a paired D. paradoxum vertically from the gills. There were significant differences (P<0.05, Tukey test) between the widths of clamp openings in D. paradoxum treated in three different solutions: the widest clamp openings were observed in the monogeneans treated in 100 mmol l(-1) potassium chloride solution (58.26±13.44 μm), followed by those treated in 20 mmol l(-1) magnesium chloride solution (37.91±7.58 μm), and finally those treated in filtered lake water (20.16±8.63 μm). This suggests that the closing of the clamps is probably not due to the continuous contraction of extrinsic muscles but is caused by the elasticity of the clamp material and that muscle activity is required for clamp opening.
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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a function for the ψ subunit in loading the escherichia coli dna polymerase sliding clamp
Journal of Biological Chemistry, 2007Co-Authors: Stephen G Anderson, Mike Odonnell, Christopher R Williams, Linda B BloomAbstract:Abstract Crystal structures of an Escherichia coli clamp loader have provided insight into the mechanism by which this molecular machine assembles ring-shaped sliding clamps onto DNA. The contributions made to the clamp loading reaction by two subunits, χ and ψ, which are not present in the crystal structures, were determined by measuring the activities of three forms of the clamp loader, γ3δδ′, γ3δδ′ψ, and γ3δδ′ψχ. The ψ subunit is important for stabilizing an ATP-induced conformational state with high affinity for DNA, whereas the χ subunit does not contribute directly to clamp loading in our assays lacking single-stranded DNA-binding protein. The ψ subunit also increases the affinity of the clamp loader for the clamp in assays in which ATPγS is substituted for ATP. Interestingly, the affinity of the γ3δδ′ complex for β is no greater in the presence than in the absence of ATPγS. A role for ψ in stabilizing or promoting ATP- and ATPγS-induced conformational changes may explain why large conformational differences were not seen in γ3δδ′ structures with and without bound ATPγS. The β clamp partially compensates for the activity of ψ when this subunit is not present and possibly serves as a scaffold on which the clamp loader adopts the appropriate conformation for DNA binding and clamp loading. Results from our work and others suggest that the ψ subunit may introduce a temporal order to the clamp loading reaction in which clamp binding precedes DNA binding.
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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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crystal structure of a dna polymerase sliding clamp from a gram positive bacterium
BMC Structural Biology, 2006Co-Authors: Maria A Argiriadi, Mike Odonnell, John Kuriyan, Eric R. Goedken, Irina BruckAbstract:Background Sliding DNA clamps are processivity factors that are required for efficient DNA replication. DNA polymerases maintain proximity to nucleic acid templates by interacting with sliding clamps that encircle DNA and thereby link the polymerase enzyme to the DNA substrate. Although the structures of sliding clamps from Gram-negative bacteria (E. coli), eukaryotes, archaea, and T4-like bacteriophages are well-known, the structure of a sliding clamp from Gram-positive bacteria has not been reported previously.
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out of plane motions in open sliding clamps molecular dynamics simulations of eukaryotic and archaeal proliferating cell nuclear antigen
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Steven L. Kazmirski, Mike Odonnell, John Kuriyan, Gregory D. Bowman, Yanxiang ZhaoAbstract:Sliding clamps are ring-like multimeric proteins that encircle duplex DNA and serve as mobile DNA-bound platforms that are essential for efficient DNA replication and repair. Sliding clamps are placed on DNA by clamp loader complexes, in which the clamp-interacting elements are organized in a right-handed spiral assembly. To understand how the flat, ring-like clamps might interact with the spiral interaction surface of the clamp loader complex, we have performed molecular dynamics simulations of sliding clamps (proliferating cell nuclear antigen from the budding yeast, humans, and an archaeal species) in which we have removed one of the three subunits so as to release the constraint of ring closure. The simulations reveal significant structural fluctuations corresponding to lateral opening and out-of-plane distortions of the clamp, which result principally from bending and twisting of the β-sheets that span the intermolecular interfaces, with smaller but similar contributions from β-sheets that span the intramolecular interfaces within each subunit. With the integrity of these β-sheets intact, the predominant fluctuations seen in the simulations are oscillations between lateral openings and right-handed spirals. The tendency for clamps to adopt a right-handed spiral conformation implies that once opened, the conformation of the clamp can easily match the spiraling of clamp loader subunits, a feature that is intrinsic to the recognition of DNA and subsequent hydrolysis of ATP by the clamp-bound clamp loader complex.