E. coli clamp loader with a closed clamp
Ontology highlight
ABSTRACT:
SUBMITTER: Zhi-Qiang Xu
PROVIDER: EMPIAR-12052 | biostudies-other |
REPOSITORIES: biostudies-other
Publications
Structural characterisation of the complete cycle of sliding clamp loading in Escherichia coli.
Xu Zhi-Qiang ZQ Jergic Slobodan S Lo Allen T Y ATY Pradhan Alok C AC Brown Simon H J SHJ Bouwer James C JC Ghodke Harshad H Lewis Peter J PJ Tolun Gökhan G Oakley Aaron J AJ Dixon Nicholas E NE
Nature communications 20240927 1
Ring-shaped DNA sliding clamps are essential for DNA replication and genome maintenance. Clamps need to be opened and chaperoned onto DNA by clamp loader complexes (CLCs). Detailed understanding of the mechanisms by which CLCs open and place clamps around DNA remains incomplete. Here, we present a series of six structures of the Escherichia coli CLC bound to an open or closed clamp prior to and after binding to a primer-template DNA, representing the most significant intermediates in the clamp l ...[more]
Similar Datasets
Project description: Not available
| EMPIAR-12095 | biostudies-other
Project description: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.
| S-EPMC3234947 | biostudies-literature
Project description:Single-stranded DNA binding proteins (SSBs) avidly bind ssDNA and yet enzymes that need to act during DNA replication and repair are not generally impeded by SSB, and are often stimulated by SSB. Here, the effects of Escherichia coli SSB on the activities of the DNA polymerase processivity clamp loader were investigated. SSB enhances binding of the clamp loader to DNA by increasing the lifetime on DNA. Clamp loading was measured on DNA substrates that differed in length of ssDNA overhangs to permit SSB binding in different binding modes. Even though SSB binds DNA adjacent to single-stranded/double-stranded DNA junctions where clamps are loaded, the rate of clamp loading on DNA was not affected by SSB on any of the DNA substrates. Direct measurements of the relative timing of DNA-SSB remodeling and enzyme-DNA binding showed that the clamp loader rapidly remodels SSB on DNA such that SSB has little effect on DNA binding rates. However, when SSB was mutated to reduce protein-protein interactions with the clamp loader, clamp loading was inhibited by impeding binding of the clamp loader to DNA. Thus, protein-protein interactions between the clamp loader and SSB facilitate rapid DNA-SSB remodeling to allow rapid clamp loader-DNA binding and clamp loading.
| S-EPMC9825162 | biostudies-literature
Project description: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.
| S-EPMC3281585 | biostudies-literature
Project description:Multi-protein clamp loader complexes are required to load sliding clamps onto DNA. In Escherichia coli the clamp loader contains three DnaX (tau/gamma) proteins, delta, and delta', which together form an asymmetric pentameric ring that also interacts with psichi. Here we used mass spectrometry to examine the assembly and dynamics of the clamp loader complex. We find that gamma exists exclusively as a stable homotetramer, while tau is in a monomer-dimer-trimer-tetramer equilibrium. delta' plays a direct role in the assembly as a tau/gamma oligomer breaker, thereby facilitating incorporation of lower oligomers. With delta', both delta and psichi stabilize the trimeric form of DnaX, thus completing the assembly. When tau and gamma are present simultaneously, mimicking the situation in vivo, subunit exchange between tau and gamma tetramers occurs rapidly to form heterocomplexes but is retarded when deltadelta' is present. The implications for intracellular assembly of the DNA polymerase III holoenzyme are discussed.
| S-EPMC4869865 | biostudies-literature
Project description: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 ATPgammaS (at a resolution of 3.5 A) and ADP (at a resolution of 4.1 A). 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 ATPgammaS 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.
| S-EPMC529418 | biostudies-literature
Project description:The eukaryotic RFC clamp loader couples the energy of ATP hydrolysis to open and close the circular PCNA sliding clamp onto primed sites for use by DNA polymerases and repair factors. Structural studies reveal clamp loaders to be heteropentamers. Each subunit contains a region of homology to AAA+ proteins that defines two domains. The AAA+ domains form a right-handed spiral upon binding ATP. This spiral arrangement generates a DNA binding site within the center of RFC. DNA enters the central chamber through a gap between the AAA+ domains of two subunits. Specificity for a primed template junction is achieved by a third domain that blocks DNA, forcing it to bend sharply. Thus only DNA with a flexible joint can bind the central chamber. DNA entry also requires a slot in the PCNA clamp, which is opened upon binding the AAA+ domains of the clamp loader. ATP hydrolysis enables clamp closing and ejection of RFC, completing the clamp loading reaction.
| S-EPMC3711244 | biostudies-literature
Project description:The Escherichia coli clamp loader, gamma complex (gamma(3)deltadelta'lambdapsi), catalyzes ATP-driven assembly of beta clamps onto primer-template DNA (p/tDNA), enabling processive replication. The mechanism by which gamma complex targets p/tDNA for clamp assembly is not resolved. According to previous studies, charged/polar amino acids inside the clamp loader chamber interact with the double-stranded (ds) portion of p/tDNA. We find that dsDNA, not ssDNA, can trigger a burst of ATP hydrolysis by gamma complex and clamp assembly, but only at far higher concentrations than p/tDNA. Thus, contact between gamma complex and dsDNA is necessary and sufficient, but not optimal, for the reaction, and additional contacts with p/tDNA likely facilitate its selection as the optimal substrate for clamp assembly. We investigated whether a conserved sequence-HRVW(279)QNRR--in delta subunit contributes to such interactions, since Tryptophan-279 specifically cross-links to the primer-template junction. Mutation of delta-W279 weakens gamma complex binding to p/tDNA, hampering its ability to load clamps and promote proccessive DNA replication, and additional mutations in the sequence (delta-R277, delta-R283) worsen the interaction. These data reveal a novel location in the C-terminal domain of the E. coli clamp loader that contributes to DNA binding and helps define p/tDNA as the preferred substrate for the reaction.
| S-EPMC2425476 | biostudies-literature
Project description:The E. coli γ clamp loader is a pentameric complex of δ, δ' and three γ subunits that opens and loads β-clamp proteins onto DNA in an ATP-dependent process essential for efficient DNA replication. ATP binding to the γ subunits promotes conformational changes that enable the clamp loader to bind and open the ring-shaped β-clamp homodimer. Here we report the nearly complete backbone and side-chain 1H, 13C and 15N NMR resonance assignments of the 242-residue truncated γ subunit of the clamp loader complex, which includes the N-terminal mini (domain I) and lid (domain II) domains. This construct represents the nucleotide binding module in the clamp loader complex and provides a model system for studies of conformational rearrangements of the clamp loader induced by nucleotide binding.
| S-EPMC8460709 | biostudies-literature
Project description:We have investigated the communication between subunits in replication factor C (RFC) from Archaeoglobus fulgidus. Mutation of the proposed arginine finger in the small subunits results in a complex that can still bind ATP but has impaired clamp-loading activity, a process that normally only requires binding of nucleotide. The small subunit alone forms a hexameric ring that is six-fold symmetric in the absence of ATP. However, this symmetry is broken when the nucleotide is bound to the complex. A conformational change associated with nucleotide binding may relate to the opening of PCNA rings by RFC during the loading reaction. The structures also reveal the importance of the N-terminal helix of each subunit at the ATP-binding site. Analysis of mutant protein complexes containing subunits lacking this N-terminal helix reveals key distinct regulatory roles during clamp loading that are different for the large and small subunits in the RFC complex.
| S-EPMC1462970 | biostudies-literature