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

ABSTRACT: 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.

SUBMITTER: Park AY

PROVIDER: S-EPMC4869865 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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

Park Ah Young AY Jergic Slobodan S Politis Argyris A Ruotolo Brandon T BT Hirshberg Daniel D Jessop Linda L LL Beck Jennifer L JL Barsky Daniel D O'Donnell Mike M Dixon Nicholas E NE Robinson Carol V CV

Structure (London, England : 1993) 20100301 3

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 ...[more]

PMID: 20223211

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Project description:Sliding clamp proteins topologically encircle DNA and play vital roles in coordinating the actions of various DNA replication, repair, and damage tolerance proteins. At least three distinct surfaces of the Escherichia coli beta clamp interact physically with the DNA that it topologically encircles. We utilized mutant beta clamp proteins bearing G66E and G174A substitutions (beta159), affecting the single-stranded DNA-binding region, or poly-Ala substitutions in place of residues 148-HQDVR-152 (beta(148-152)), affecting the double-stranded DNA binding region, to determine the biological relevance of clamp-DNA interactions. As part of this work, we solved the X-ray crystal structure of beta(148-152), which verified that the poly-Ala substitutions failed to significantly alter the tertiary structure of the clamp. Based on functional assays, both beta159 and beta(148-152) were impaired for loading and retention on a linear primed DNA in vitro. In the case of beta(148-152), this defect was not due to altered interactions with the DnaX clamp loader, but rather was the result of impaired beta(148-152)-DNA interactions. Once loaded, beta(148-152) was proficient for DNA polymerase III (Pol III) replication in vitro. In contrast, beta(148-152) was severely impaired for Pol II and Pol IV replication and was similarly impaired for direct physical interactions with these Pols. Despite its ability to support Pol III replication in vitro, beta(148-152) was unable to support viability of E. coli. Nevertheless, physiological levels of beta(148-152) expressed from a plasmid efficiently complemented the temperature-sensitive growth phenotype of a strain expressing beta159 (dnaN159), provided that Pol II and Pol IV were inactivated. Although this strain was impaired for Pol V-dependent mutagenesis, inactivation of Pol II and Pol IV restored the Pol V mutator phenotype. Taken together, these results support a model in which a sophisticated combination of competitive clamp-DNA, clamp-partner, and partner-DNA interactions serve to manage the actions of the different E. coli Pols in vivo.

Dataset Information

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

Publications

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

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