Dimer of the .beta. subunit of Escherichia coli DNA polymerase III holoenzyme is dissociated into monomers upon binding magnesium(II)

Griep, Mark A.; McHenry, Charles S.

doi:10.1021/bi00414a040

Cited by 25 publications

(16 citation statements)

References 27 publications

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“…This Cys residue is located on the opposite face of ␤ to which the ␥ complex and core polymerase bind (1,29). Control experiments in our laboratory demonstrated that labeling ␤ did not affect its activity in replication assays (data not shown), as has been reported by others (28,30). The anisotropy of pyrene increased when the ␥ complex bound ␤ and decreased its rate of rotational diffusion.…”

Section: Kinetics Of Atp Hydrolysis By the Wild-type ␥ Complex And Srcsupporting

confidence: 71%

“…An anisotropybased assay in which ␤ was covalently labeled with N-(1-pyrenyl)maleimide was used to measure these protein-protein interactions. Previous studies in another laboratory have demonstrated that ␤ is specifically labeled at one of its four cysteines (Cys-333) with thiol-reactive probes, including pyrenylmaleimide (28). This Cys residue is located on the opposite face of ␤ to which the ␥ complex and core polymerase bind (1,29).…”

Section: Kinetics Of Atp Hydrolysis By the Wild-type ␥ Complex And Srcmentioning

confidence: 99%

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Mechanism of Loading the Escherichia coli DNA Polymerase III Sliding Clamp

Snyder

Williams

Johnson

et al. 2004

Journal of Biological Chemistry

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Sliding clamps tether DNA polymerases to DNA to increase the processivity of synthesis. The Escherichia coli ␥ complex loads the ␤ sliding clamp onto DNA in an ATP-dependent reaction in which ATP binding and hydrolysis modulate the affinity of the ␥ complex for ␤ and DNA. This is the second of two reports (Williams, C. R., Snyder, A. K., Kuzmič , P., O'Donnell, M., and Bloom, L. B. (2004) J. Biol. Chem. 279, 4376 -4385) addressing the question of how ATP binding and hydrolysis regulate specific interactions with DNA and ␤. Mutations were made to an Arg residue in a conserved SRC motif in the ␦ and ␥ subunits that interacts with the ATP site of the neighboring ␥ subunit. Mutation of the ␦ subunit reduced the ATP-dependent ␤ binding activity, whereas mutation of the ␥ subunits reduced the DNA binding activity of the ␥ complex. The ␥ complex containing the ␦ mutation gave a pre-steady-state burst of ATP hydrolysis, but at a reduced rate and amplitude relative to the wild-type ␥ complex. A pre-steady-state burst of ATP hydrolysis was not observed for the complex containing the ␥ mutations, consistent with the reduced DNA binding activity of this complex. The differential effects of these mutations suggest that ATP binding at the ␥ 1 site may be coupled to conformational changes that largely modulate interactions with ␤, whereas ATP binding at the ␥ 2 and/or ␥ 3 site may be coupled to conformational changes that have a major role in interactions with DNA. Additionally, these results show that the "arginine fingers" play a structural role in facilitating the formation of a conformation that has high affinity for ␤ and DNA.Sliding clamps tether DNA polymerases to their templates, enabling the polymerases to rapidly incorporate thousands of nucleotides into a growing polymer without dissociating from DNA. Crystal structures of sliding clamps from bacteria, bacteriophage, and humans have revealed a mechanism by which this is possible (1-4). Sliding clamps are composed of individual subunits that assemble into rings with a central opening large enough to encircle duplex DNA. These ring-shaped clamps must be assembled on DNA by the activity of a clamp loader that uses energy derived from ATP to power its mechanical task (reviewed in Ref. 5).In Escherichia coli, the clamp loader is composed of seven subunits, three copies of the dnaX gene product, ␦, ␦Ј, , and (6 -8). The DnaX proteins form the motor of the clamp loader, and each is capable of binding and hydrolyzing 1 molecule of ATP during the clamp loading process. In vivo, two forms of the DnaX protein are produced: a full-length gene product () and a truncated gene product (␥) with the same sequence, but only about two-thirds the length of (9 -11). The clamp loader at the replication fork most likely contains two copies of the subunit and a single copy of ␥ (7, 8). The unique C-terminal end present in interacts with other enzymes and coordinates the activities present at the replication fork, whereas the N-terminal domain shared by the and ␥ subunits provides the activ...

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Section: Kinetics Of Atp Hydrolysis By the Wild-type ␥ Complex And Srcsupporting

confidence: 71%

Section: Kinetics Of Atp Hydrolysis By the Wild-type ␥ Complex And Srcmentioning

confidence: 99%

Mechanism of Loading the Escherichia coli DNA Polymerase III Sliding Clamp

Snyder

Williams

Johnson

et al. 2004

Journal of Biological Chemistry

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“…Concentrations of ␥ complex were determined by measuring the absorbance at 280 nm in 6 M guanidine hydrochloride and using the calculated extinction coefficient (220,050 M Ϫ1 cm Ϫ1 ). The ␤ clamp was covalently labeled on Cys-299 with N-(1-pyrene)maleimide (Molecular Probes) and purified as described previously (27,28). The protein concentration of ␤ PY was determined using the modified Lowry protein assay (Pierce).…”

Section: Methodsmentioning

confidence: 99%

A Slow ATP-induced Conformational Change Limits the Rate of DNA Binding but Not the Rate of β Clamp Binding by the Escherichia coli γ Complex Clamp Loader

Thompson

Paschall

O'Donnell

et al. 2009

Journal of Biological Chemistry

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In Escherichia coli, the ␥ complex clamp loader loads the ␤-sliding clamp onto DNA. The ␤ clamp tethers DNA polymerase III to DNA and enhances the efficiency of replication by increasing the processivity of DNA synthesis. In the presence of ATP, ␥ complex binds ␤ and DNA to form a ternary complex. Binding to primed template DNA triggers ␥ complex to hydrolyze ATP and release the clamp onto DNA. Here, we investigated the kinetics of forming a ternary complex by measuring rates of ␥ complex binding ␤ and DNA. A fluorescence intensity-based ␤ binding assay was developed in which the fluorescence of pyrene covalently attached to ␤ increases when bound by ␥ complex. Using this assay, an association rate constant of 2.3 ؋ 10 7 M ؊1 s ؊1 for ␥ complex binding ␤ was determined. The rate of ␤ binding was the same in experiments in which ␥ complex was preincubated with ATP before adding ␤ or added directly to ␤ and ATP. In contrast, when ␥ complex is preincubated with ATP, DNA binding is faster than when ␥ complex is added to DNA and ATP at the same time. Slow DNA binding in the absence of ATP preincubation is the result of a rate-limiting ATP-induced conformational change. Our results strongly suggest that the ATP-induced conformational changes that promote ␤ binding and DNA binding differ. The slow ATP-induced conformational change that precedes DNA binding may provide a kinetic preference for ␥ complex to bind ␤ before DNA during the clamp loading reaction cycle.Two accessory factors, a sliding clamp and a clamp loader, enhance the efficiency of DNA replication by increasing the processivity of DNA synthesis. The clamp loader assembles clamps onto DNA. The clamp binds the DNA polymerase to increase the processivity of DNA synthesis from tens to thousands of nucleotides in a single binding event. These processivity factors are conserved from bacteria to humans (reviewed in Refs. 1 and 2). The Escherichia coli ␤ clamp is composed of two identical protein monomers assembled into a ring-shaped dimer, which encircles duplex DNA (3, 4). The clamp slides freely along the DNA while tethering DNA polymerase III to the DNA template. The E. coli clamp loader is composed of seven subunits. At the replication fork, a complete clamp loader includes three copies of the dnaX gene product and one copy each of ␦, ␦Ј, , and (5-7). The dnaX gene produces two proteins, a full-length protein () and a truncated protein (␥), which is created by a translational frameshift (8 -10). Clamp loaders containing either three copies of the subunit ( complex, 3 ␦␦Ј) or three copies of the ␥ subunit (␥ complex, ␥ 3 ␦␦Ј) are fully active in clamp loading (6). The subunits have an additional C-terminal extension that coordinates the activities of the replisome (reviewed in Refs. 11 and 12). The clamp loader used in all experiments in this study is the ␥ complex.In the presence of ATP, ␥ complex binds with high affinity to ␤ and DNA to form a ternary complex. Binding to primed template DNA triggers the clamp loader to hydrolyze all three molecules of ATP (...

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“…N-terminal biotinylated 20-mer peptides were diluted in PBS (30 l) Fluorescence Measurements. The ␤ subunit can be uniquely labeled at Cys-333 by using maleimide derivatives (18). ␤ (3 mg) was labeled by using Oregon Green 488 maleimide (Molecular Probes) in 1 ml of 50 mM potassium phosphate (pH 7.5) and 100 mM NaCl.…”

Section: Methodsmentioning

confidence: 99%

A peptide switch regulates DNA polymerase processivity

Saro

Georgescu

O'Donnell

2003

Proc. Natl. Acad. Sci. U.S.A.

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Chromosomal DNA polymerases are tethered to DNA by a circular sliding clamp for high processivity. However, lagging strand synthesis requires the polymerase to rapidly dissociate on finishing each Okazaki fragment. The Escherichia coli replicase contains a subunit ( ) that promotes separation of polymerase from its clamp on finishing DNA segments. This report reveals the mechanism of this process. We find that binds the C-terminal residues of the DNA polymerase. Surprisingly, this same C-terminal ''tail'' of the polymerase interacts with the ␤ clamp, and competes with ␤ for this sequence. Moreover, acts as a DNA sensor. On binding primed DNA, releases the polymerase tail, allowing polymerase to bind ␤ for processive synthesis. But on sensing the DNA is complete (duplex), sequesters the polymerase tail from ␤, disengaging polymerase from DNA. Therefore, DNA sensing by switches the polymerase peptide tail on and off the clamp and coordinates the dynamic turnover of polymerase during lagging strand synthesis.

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Dimer of the .beta. subunit of Escherichia coli DNA polymerase III holoenzyme is dissociated into monomers upon binding magnesium(II)

Cited by 25 publications

References 27 publications

Mechanism of Loading the Escherichia coli DNA Polymerase III Sliding Clamp

Mechanism of Loading the Escherichia coli DNA Polymerase III Sliding Clamp

A Slow ATP-induced Conformational Change Limits the Rate of DNA Binding but Not the Rate of β Clamp Binding by the Escherichia coli γ Complex Clamp Loader

A peptide switch regulates DNA polymerase processivity

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