Mechanism of Loading the Escherichia coli DNA Polymerase III Sliding Clamp

Williams, Christopher R.; Snyder, Anita K.; Kuzmič, Petr; O’Donnell, Michael; Bloom, Linda B.

doi:10.1074/jbc.m310429200

Cited by 37 publications

(35 citation statements)

References 46 publications

(96 reference statements)

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“…The ITC results are consistent with recent time-resolved measurements of interactions between the E. coli clamp loader, clamp, and ATP, which can be interpreted in terms of the binding of two molecules of ATP to the clamp loader in the absence of clamp, with the stoichiometry of ATP binding increasing to 3 in the presence of the clamp (39). Analysis of an archaeal clamp-loader complex with four potential ATP-binding sites suggests that only two ATP molecules are bound in the absence of the clamp, although four are bound in the presence of clamp and DNA (40).…”

Section: Resultssupporting

confidence: 77%

Structural analysis of the inactive state of the Escherichia coli DNA polymerase clamp-loader complex

Kazmirski

Podobnik

Weitze

et al. 2004

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

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Clamp-loader complexes are heteropentameric AAA ؉ ATPases that load sliding clamps onto DNA. The structure of the nucleotide-free Escherichia coli clamp loader had been determined previously and led to the proposal that the clamp-loader cycles between an inactive state, in which the ATPase domains form a closed ring, and an active state that opens up to form a ''C'' shape. The crystal structure was interpreted as being closer to the active state than the inactive state. The crystal structure of a nucleotide-bound eukaryotic clamp loader [replication factor C (RFC)] revealed a different and more tightly packed spiral organization of the ATPase domains, raising questions about the significance of the conformation seen earlier for the bacterial clamp loader. We describe crystal structures of the E. coli clamp-loader complex bound to the ATP analog ATP␥S (at a resolution of 3.5 Å) and ADP (at a resolution of 4.1 Å). These structures are similar to that of the nucleotide-free clamp-loader complex. Only two of the three functional ATP-binding sites are occupied by ATP␥S or ADP in these structures, and the bound nucleotides make no interfacial contacts in the complex. These results, along with data from isothermal titration calorimetry, molecular dynamics simulations, and comparison with the RFC structure, suggest that the more open form of the E. coli clamp loader described earlier and in the present work corresponds to a stable inactive state of the clamp loader in which the ATPase domains are prevented from engaging the clamp in the highly cooperative manner seen in the fully ATP-loaded RFC-clamp structure.AAA ϩ ATPase ͉ clamp loader ͉ DNA polymerase III ͉ DNA replication ͉ replication factor C C hromosomal replicases in all of the branches of life achieve high processivity by tethering their catalytic subunits to sliding DNA clamps (1-3). The closed circular sliding clamps are loaded onto DNA by ATP-dependent clamp-loader complexes (4), which are conserved heteropentameric ATPase assemblies. Most sliding clamps are stable closed rings in solution, although recent studies suggest that the clamp in T4 bacteriophage may be open in solution and that the role of the clamp loader might be to stabilize the open form and guide it to DNA (5). The subunits of the clamp-loader complexes consist of three conserved domains, with the first two being closely related to the nucleotide-binding domains of AAA ϩ ATPases (6-10). The AAA ϩ domains of the clamp loader are suspended loosely from a circular helical collar that is formed by the third (C-terminal) domains.ATP binding to the clamp loader triggers a conformational change that allows the complex to bind to and open the clamp and load it onto DNA (11, 12). In an intriguing control mechanism, the interaction with DNA stimulates the otherwise suppressed ATPase activity of the clamp loader, leading to ATP hydrolysis (11, 13) and separation of the clamp-loader complex from the DNA-loaded clamp. How ATP binding is coupled to loading of the clamp on DNA is poorly understood, and eluc...

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Section: Resultssupporting

confidence: 77%

Structural analysis of the inactive state of the Escherichia coli DNA polymerase clamp-loader complex

Kazmirski

Podobnik

Weitze

et al. 2004

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

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“…The sequence of the primed template differs from that in the accompanying article (37), but we have demonstrated that pre-steady-state kinetics of clamp loading and ATP hydrolysis are not affected by differences in sequence (data not shown). For anisotropy experiments, the single-stranded 50-mer and 105-mer template were covalently labeled with X-rhodamine isothiocyanate (RhX) at the 5Ј-end as described in the accompanying article (37).…”

Section: Methodsmentioning

confidence: 81%

“…Samples were excited with vertically polarized light, and emission of vertically (I VV ) and horizontally (I VH ) polarized light was measured simultaneously in a T-format. Anisotropy (r) was calculated from polarized intensities as described in the accompanying article (37). There were no changes in the total intensities of RhX and pyrene as a function of the ␥ complex concentration, indicating that there were no changes in effective quantum yields during the course of the experiment.…”

Section: Methodsmentioning

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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“…Analysis of an active assembly from E. coli revealed the overall architecture of the loader (19) and also provided a context with which to interpret the distinct roles suggested for its three nucleotide binding sites (20)(21)(22). A (23)] in complex with a monomeric form of the bacterial clamp (24) demonstrated how the closed ring might be opened (25).…”

mentioning

confidence: 99%

The opened processivity clamp slides into view

Jeruzalmi¹

2005

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

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Mechanism of Loading the Escherichia coli DNA Polymerase III Sliding Clamp

Cited by 37 publications

References 46 publications

Structural analysis of the inactive state of the Escherichia coli DNA polymerase clamp-loader complex

Structural analysis of the inactive state of the Escherichia coli DNA polymerase clamp-loader complex

Mechanism of Loading the Escherichia coli DNA Polymerase III Sliding Clamp

The opened processivity clamp slides into view

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