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      The C-Terminal Domain of the Bacterial SSB Protein Acts as a DNA Maintenance Hub at Active Chromosome Replication Forks

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          Abstract

          We have investigated in vivo the role of the carboxy-terminal domain of the Bacillus subtilis Single-Stranded DNA Binding protein (SSB Cter) as a recruitment platform at active chromosomal forks for many proteins of the genome maintenance machineries. We probed this SSB Cter interactome using GFP fusions and by Tap-tag and biochemical analysis. It includes at least 12 proteins. The interactome was previously shown to include PriA, RecG, and RecQ and extended in this study by addition of DnaE, SbcC, RarA, RecJ, RecO, XseA, Ung, YpbB, and YrrC. Targeting of YpbB to active forks appears to depend on RecS, a RecQ paralogue, with which it forms a stable complex. Most of these SSB partners are conserved in bacteria, while others, such as the essential DNA polymerase DnaE, YrrC, and the YpbB/RecS complex, appear to be specific to B. subtilis. SSB Cter deletion has a moderate impact on B. subtilis cell growth. However, it markedly affects the efficiency of repair of damaged genomic DNA and arrested replication forks. ssbΔCter mutant cells appear deficient in RecA loading on ssDNA, explaining their inefficiency in triggering the SOS response upon exposure to genotoxic agents. Together, our findings show that the bacterial SSB Cter acts as a DNA maintenance hub at active chromosomal forks that secures their propagation along the genome.

          Author Summary

          Cell multiplication relies primarily on the complete and accurate duplication of the genome. Thus, all organisms have evolved multiple mechanisms to protect, repair, and re-activate the DNA replication forks. A large body of research is currently aimed at deciphering the mechanisms that precisely direct the proteins involved in these rescue pathways towards the chromosome replication forks. Here, we have used the model bacterium Bacillus subtilis to demonstrate that the active chromosomal DNA replication forks are pre-equipped with many such rescue effectors via their direct physical interaction with the carboxy-terminal end (Cter) of the Single-Stranded DNA Binding protein (SSB). A detailed analysis of the multiple defects of viable B. subtilis mutants deleted for the Cter of SSB (SSB Cter) revealed the vital role of this domain for the maintenance of genome integrity and fork propagation. The inability to grow at high temperature is a major defect of the ssbΔCter mutant. We show that this lethality can be specifically suppressed by overexpression of RecO, one of the numerous partners of SSB, apparently by mediating the loading of the RecA recombinase on ssDNA.

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          A generic protein purification method for protein complex characterization and proteome exploration.

          We have developed a generic procedure to purify proteins expressed at their natural level under native conditions using a novel tandem affinity purification (TAP) tag. The TAP tag allows the rapid purification of complexes from a relatively small number of cells without prior knowledge of the complex composition, activity, or function. Combined with mass spectrometry, the TAP strategy allows for the identification of proteins interacting with a given target protein. The TAP method has been tested in yeast but should be applicable to other cells or organisms.
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            SSB as an organizer/mobilizer of genome maintenance complexes.

            When duplex DNA is altered in almost any way (replicated, recombined, or repaired), single strands of DNA are usually intermediates, and single-stranded DNA binding (SSB) proteins are present. These proteins have often been described as inert, protective DNA coatings. Continuing research is demonstrating a far more complex role of SSB that includes the organization and/or mobilization of all aspects of DNA metabolism. Escherichia coli SSB is now known to interact with at least 14 other proteins that include key components of the elaborate systems involved in every aspect of DNA metabolism. Most, if not all, of these interactions are mediated by the amphipathic C-terminus of SSB. In this review, we summarize the extent of the eubacterial SSB interaction network, describe the energetics of interactions with SSB, and highlight the roles of SSB in the process of recombination. Similar themes to those highlighted in this review are evident in all biological systems.
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              Three-dimensional structure of the β subunit of E. coli DNA polymerase III holoenzyme: A sliding DNA clamp

              The crystal structure of the beta subunit (processivity factor) of DNA polymerase III holoenzyme has been determined at 2.5 A resolution. A dimer of the beta subunit (M(r) = 2 x 40.6 kd, 2 x 366 amino acid residues) forms a ring-shaped structure lined by 12 alpha helices that can encircle duplex DNA. The structure is highly symmetrical, with each monomer containing three domains of identical topology. The charge distribution and orientation of the helices indicate that the molecule functions by forming a tight clamp that can slide on DNA, as shown biochemically. A potential structural relationship is suggested between the beta subunit and proliferating cell nuclear antigen (PCNA, the eukaryotic polymerase delta [and epsilon] processivity factor), and the gene 45 protein of the bacteriophage T4 DNA polymerase.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, USA )
                1553-7390
                1553-7404
                December 2010
                December 2010
                9 December 2010
                : 6
                : 12
                : e1001238
                Affiliations
                [1 ]Laboratoire de Microbiologie et Génétique Moléculaires, Université de Toulouse, Centre National de la Recherche Scientifique, LMGM-UMR5100, Toulouse, France
                [2 ]INRA, UMR1319 Micalis (Microbiologie de l'Alimentation au service de la Santé), Domaine de Vilvert, Jouy-en-Josas, France
                [3 ]Institut de Biochimie et de Biophysique Moléculaire et Cellulaire, Université de Paris-Sud, Centre National de la Recherche Scientifique, UMR8619, IFR115, Orsay, France
                Université Paris Descartes, INSERM U571, France
                Author notes

                Conceived and designed the experiments: AC FL SQC PP. Performed the experiments: AC FL SM SQC PP. Analyzed the data: AC FL SM SQC PP. Contributed reagents/materials/analysis tools: PP. Wrote the paper: FL PP.

                Article
                10-PLGE-RA-3271R3
                10.1371/journal.pgen.1001238
                3000357
                21170359
                7a605311-470f-4924-93ca-00bd94b3d6f1
                Costes et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 24 May 2010
                : 4 November 2010
                Page count
                Pages: 15
                Categories
                Research Article
                Genetics and Genomics/Chromosome Biology
                Molecular Biology/DNA Repair
                Molecular Biology/DNA Replication
                Molecular Biology/Recombination

                Genetics
                Genetics

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