7
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      Discovery of a dual protease mechanism that promotes DNA damage checkpoint recovery

      research-article

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          The DNA damage response is a signaling pathway found throughout biology. In many bacteria the DNA damage checkpoint is enforced by inducing expression of a small, membrane bound inhibitor that delays cell division providing time to repair damaged chromosomes. How cells promote checkpoint recovery after sensing successful repair is unknown. By using a high-throughput, forward genetic screen, we identified two unrelated proteases, YlbL and CtpA, that promote DNA damage checkpoint recovery in Bacillus subtilis. Deletion of both proteases leads to accumulation of the checkpoint protein YneA. We show that DNA damage sensitivity and increased cell elongation in protease mutants depends on yneA. Further, expression of YneA in protease mutants was sufficient to inhibit cell proliferation. Finally, we show that both proteases interact with YneA and that one of the two proteases, CtpA, directly cleaves YneA in vitro. With these results, we report the mechanism for DNA damage checkpoint recovery in bacteria that use membrane bound cell division inhibitors.

          Author summary

          Prokaryotes and eukaryotes coordinate cell division to genome integrity using DNA damage checkpoints. Many bacteria express a small, membrane binding protein to slow cell division when obstacles to DNA replication are encountered. Cell division inhibitors of this class have been identified in several bacterial species, yet the mechanism used to alleviate inhibition has remained unknown. Using forward genetics, we identified two unstudied genes, coding for the proteases YlbL and CtpA, that when deleted result in sensitivity to drugs that directly damage DNA. We show that sensitivity to DNA damage in protease mutants is a result of accumulation of the cell division inhibitor. Further, we show that YlbL and CtpA are responsible for degrading the cell division inhibitor allowing for cell division to resume. Importantly, these two proteases are not homologs, demonstrating a striking example of a bacterium using non-homologous enzymes to degrade a single substrate. Our investigation uncovers the previously unknown mechanism used to remove a cell division inhibitor, while also illuminating a potential strategy that bacteria can use to regulate signaling pathways. The use of multiple, unrelated proteins to perform a single function may represent a strategy employed throughout biological systems.

          Related collections

          Most cited references62

          • Record: found
          • Abstract: not found
          • Article: not found

          Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing

            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            Tn-seq; high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms

            Biological pathways are structured in complex networks of interacting genes. Solving the architecture of such networks may provide valuable information, such as how microorganisms cause disease. Here we present a method (Tn-seq) for accurately determining quantitative genetic interactions on a genome-wide scale in microorganisms. Tn-seq is based on the assembly of a saturated Mariner transposon insertion library. After library selection, changes in frequency of each insertion mutant are determined by sequencing of the flanking regions en masse. These changes are used to calculate each mutant’s fitness. Fitness was determined for each gene of the gram-positive bacterium Streptococcus pneumoniae, a causative agent of pneumonia and meningitis. A genome-wide screen for genetic interactions identified both alleviating and aggravating interactions that could be further divided into seven distinct categories. Due to the wide activity of the Mariner transposon, Tn-seq has the potential to contribute to the exploration of complex pathways across many different species.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms.

              Our knowledge of gene function has increasingly lagged behind gene discovery, hindering our understanding of the genetic basis of microbial phenotypes. Recently, however, massively parallel sequencing has been combined with traditional transposon mutagenesis in techniques referred to as transposon sequencing (Tn-seq), high-throughput insertion tracking by deep sequencing (HITS), insertion sequencing (INSeq) and transposon-directed insertion site sequencing (TraDIS), making it possible to identify putative gene functions in a high-throughput manner. Here, we describe the similarities and differences of these related techniques and discuss their application to the probing of gene function and higher-order genome organization.
                Bookmark

                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ResourcesRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Formal analysisRole: InvestigationRole: Methodology
                Role: Data curationRole: Formal analysis
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: Project administrationRole: ResourcesRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS Genet
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, CA USA )
                1553-7390
                1553-7404
                6 July 2018
                July 2018
                : 14
                : 7
                : e1007512
                Affiliations
                [001]Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI, United States of America
                Université Paris Descartes, INSERM U1001, FRANCE
                Author notes

                The authors have declared that no competing interests exist.

                [¤]

                Current address: Department of Bacteriology, University of Wisconsin–Madison, Madison, WI, United States of America

                Author information
                http://orcid.org/0000-0001-6404-5652
                http://orcid.org/0000-0002-9600-7623
                Article
                PGENETICS-D-18-00936
                10.1371/journal.pgen.1007512
                6051672
                29979679
                da317708-e649-4863-a663-207aeec809ab
                © 2018 Burby 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
                : 8 May 2018
                : 23 June 2018
                Page count
                Figures: 7, Tables: 1, Pages: 26
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100000057, National Institute of General Medical Sciences;
                Award ID: GM107312
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000057, National Institute of General Medical Sciences;
                Award ID: T32 GM007544
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000082, Division of Graduate Education;
                Award ID: DGE 1256260
                Award Recipient :
                Funded by: University of Michigan College of Literature, Sciences and the Arts
                Award Recipient :
                This work was supported by National Institutes of Health ( https://www.nigms.nih.gov/Pages/default.aspx) grant R01 GM107312 to LAS. JWS was supported in part by National Institutes of Health ( https://www.nigms.nih.gov/Pages/default.aspx) T32 GM007544 and Associate Professor Funds from the College of Literature, Science and the Arts at the University of Michigan ( https://lsa.umich.edu) to LAS. PEB was supported by a pre-doctoral fellowship from the National Science Foundation (Division of Graduate Education 1256260). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Biochemistry
                Enzymology
                Enzymes
                Proteases
                Biology and Life Sciences
                Biochemistry
                Proteins
                Enzymes
                Proteases
                Biology and life sciences
                Genetics
                DNA
                DNA damage
                Biology and life sciences
                Biochemistry
                Nucleic acids
                DNA
                DNA damage
                Biology and Life Sciences
                Cell Biology
                Cell Processes
                Cell Cycle and Cell Division
                Biology and Life Sciences
                Organisms
                Bacteria
                Bacillus
                Bacillus Subtilis
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbial Pathogens
                Bacterial Pathogens
                Bacillus
                Bacillus Subtilis
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Pathogens
                Microbial Pathogens
                Bacterial Pathogens
                Bacillus
                Bacillus Subtilis
                Research and Analysis Methods
                Experimental Organism Systems
                Prokaryotic Models
                Bacillus Subtilis
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Cell Membranes
                Membrane Proteins
                Biology and life sciences
                Genetics
                DNA
                DNA recombination
                Homologous Recombination
                Biology and life sciences
                Biochemistry
                Nucleic acids
                DNA
                DNA recombination
                Homologous Recombination
                Biology and Life Sciences
                Anatomy
                Body Fluids
                Blood
                Blood Serum
                Immune Serum
                Medicine and Health Sciences
                Anatomy
                Body Fluids
                Blood
                Blood Serum
                Immune Serum
                Biology and Life Sciences
                Physiology
                Body Fluids
                Blood
                Blood Serum
                Immune Serum
                Medicine and Health Sciences
                Physiology
                Body Fluids
                Blood
                Blood Serum
                Immune Serum
                Biology and Life Sciences
                Genetics
                Gene Expression
                Gene Regulation
                Custom metadata
                vor-update-to-uncorrected-proof
                2018-07-18
                All relevant data are within the paper and its Supporting Information files with sequencing data available using accession number GSE109366. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE109366

                Genetics
                Genetics

                Comments

                Comment on this article