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Solution study of the Escherichia coli DNA polymerase III clamp loader reveals the location of the dynamic ?? heterodimer.

ABSTRACT: Several X-ray crystal structures of the E. coli core clamp loader containing the five core (?', ?, and three truncated ?) subunits have been determined, but they lack the ? and ? subunits. We report the first solution structure of the complete seven-subunit clamp loader complex using small angle X-ray scattering. This structure not only provides information about the location of the ? and ? subunits but also provides a model of the dynamic nature of the clamp loader complex.

SUBMITTER: Tondnevis F

PROVIDER: S-EPMC4711647 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Solution study of the Escherichia coli DNA polymerase III clamp loader reveals the location of the dynamic ψχ heterodimer.

Tondnevis Farzaneh F Gillilan Richard E RE Bloom Linda B LB McKenna Robert R

Structural dynamics (Melville, N.Y.) 20150818 5

Several X-ray crystal structures of the E. coli core clamp loader containing the five core (δ', δ, and three truncated γ) subunits have been determined, but they lack the ψ and χ subunits. We report the first solution structure of the complete seven-subunit clamp loader complex using small angle X-ray scattering. This structure not only provides information about the location of the χ and ψ subunits but also provides a model of the dynamic nature of the clamp loader complex. ...[more]

PMID: 26798827

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Escherichia coli processivity clamp ? from DNA polymerase III is dynamic in solution.

Project description:Escherichia coli DNA polymerase III is a highly processive replicase because of the presence of the ? clamp protein that tethers DNA polymerases to DNA. The ? clamp is a head-to-tail ring-shaped homodimer, in which each protomer contains three structurally similar domains. Although multiple studies have probed the functions of the ? clamp, a detailed understanding of the conformational dynamics of the ? clamp in solution is lacking. Here we used hydrogen exchange mass spectrometry to characterize the conformation and dynamics of the intact dimer ? clamp and a variant form (I272A/L273A) with a weakened ability to dimerize in solution. Our data indicate that the ? clamp is not a static closed ring but rather is dynamic in solution. The three domains exhibited different dynamics, though they share a highly similar tertiary structure. Domain I, which controls the opening of the clamp by dissociating from domain III, contained several highly flexible peptides that underwent partial cooperative unfolding (EX1 kinetics) with a half-life of ~4 h. The comparison between the ? monomer variant and the wild-type ? clamp showed that the ? monomer was more dynamic. In the monomer, partial unfolding was much faster and additional regions of domain III also underwent partial unfolding with a half-life of ~1 h. Our results suggest that the ? subunit of the clamp loader may function as a "ring holder" to stabilize the transient opening of the ? clamp, rather than as a "ring opener".

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

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The Escherichia coli clamp loader can actively pry open the β-sliding clamp.

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E. coli clamp loader with a closed clamp

Project description: Not available

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The Escherichia coli clamp loader rapidly remodels SSB on DNA to load clamps.

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.

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A single subunit directs the assembly of the Escherichia coli DNA sliding clamp loader.

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Nuclear magnetic resonance solution structure of the Escherichia coli DNA polymerase III theta subunit.

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Collision with duplex DNA renders Escherichia coli DNA polymerase III holoenzyme susceptible to DNA polymerase IV-mediated polymerase switching on the sliding clamp.

Project description:Organisms possess multiple DNA polymerases (Pols) and use each for a different purpose. One of the five Pols in Escherichia coli, DNA polymerase IV (Pol IV), encoded by the dinB gene, is known to participate in lesion bypass at certain DNA adducts. To understand how cells choose Pols when the replication fork encounters an obstacle on template DNA, the process of polymerase exchange from the primary replicative enzyme DNA polymerase III (Pol III) to Pol IV was studied in vitro. Replicating Pol III forming a tight holoenzyme (Pol III HE) with the sliding clamp was challenged by Pol IV on a primed ssDNA template carrying a short inverted repeat. A rapid and lesion-independent switch from Pol III to Pol IV occurred when Pol III HE encountered a hairpin stem duplex, implying that the loss of Pol III-ssDNA contact induces switching to Pol IV. Supporting this idea, mutant Pol III with an increased affinity for ssDNA was more resistant to Pol IV than wild-type Pol III was. We observed that an exchange between Pol III and Pol IV also occurred when Pol III HE collided with primer/template duplex. Our data suggest that Pol III-ssDNA interaction may modulate the susceptibility of Pol III HE to Pol IV-mediated polymerase exchange.

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