Structural studies on the P1 plasmid partition apparatus

Summary

Principal Investigator: Maria Schumacher
Abstract: The survival of any species demands the faithful inheritance of genetic information. Essential to this process are the directed movements and positioning of chromosomes or plasmids such that they are accurately distributed to the daughter cells at cell division. Although prokaryotes do not undergo the complex mitotic steps associated with eukaryotic cells, prokaryotic chromosomes are nevertheless dynamically arranged during the cell cycle via the action of segregation, or par systems. Coordination of this process requires precise protein-DNA and protein-protein interactions carried out by par systems. All known plasmid encoded par loci specify three components: a cis-acting centromere site (parS), and two proteins, ParB and ParA. In the E. coli P1 par system, which has served as a paradigm for understanding partition, the genes for ParA and ParB form an operon, and the approximately 74 bp partition site, parS, is located immediately downstream of parB . ParB, a 38 kDa protein with no sequence homology to any protein, is a DNA-binding protein and, along with IMF or alone, binds to a highly complex centromere-like site, parS, to form the partition complex. ParA, a 44 kDa Walker-type ATPase, utilizes the energy of ATP hydrolysis to drive plasmid separation after interacting with ParB in the partition complex. Our recent structure determinations of ParB and the minimal partition site have revealed novel DNA-binding characteristics of ParB that explain its ability to bind, spread and pair plasmids. Thus, these structures have provided unprecedented insight into the mechanism of partition complex formation. Completely unknown, however, are structural bases for plasmid separation, the step carried out by ParA. Thus, in this proposal we will build on our recent progress towards a full elucidation of P1 partition with the following Specific Aims: (1) fully elucidate the mechanism of P1 partition complex formation through structural and biochemical studies on the ParB-parS partition complex. (2) Clarify the mechanism of P1 plasmid separation via structural and biochemical studies on the key end states of P1 ParA (apoParA, ParA-ADP and the ParA(K188Q)-(AMP-PCP)-ParB(1-28) complex). These structures will provide the foundation for understanding how partition systems function to drive chromosome segregation in prokaryotes and may provide potential points of therapeutic intervention against pathogenic bacteria, which also depend on par systems for segregation and thus, survival.
Funding Period: 2006-09-28 - 2010-08-31
more information: NIH RePORT

Top Publications

  1. pmc Structures of the Bacillus subtilis glutamine synthetase dodecamer reveal large intersubunit catalytic conformational changes linked to a unique feedback inhibition mechanism
    David S Murray
    From the Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239
    J Biol Chem 288:35801-11. 2013
  2. pmc The structure of irisin reveals a novel intersubunit β-sheet fibronectin type III (FNIII) dimer: implications for receptor activation
    Maria A Schumacher
    From the Departments of Biochemistry and
    J Biol Chem 288:33738-44. 2013
  3. pmc SlmA forms a higher-order structure on DNA that inhibits cytokinetic Z-ring formation over the nucleoid
    Nam K Tonthat
    Department of Biochemistry, Duke University School of Medicine, Durham, NC 27710, USA
    Proc Natl Acad Sci U S A 110:10586-91. 2013
  4. pmc Uncoupling of nucleotide hydrolysis and polymerization in the ParA protein superfamily disrupts DNA segregation dynamics
    Aneta Dobruk-Serkowska
    University of Manchester, Manchester M1 7DN, United Kingdom
    J Biol Chem 287:42545-53. 2012
  5. ncbi Molecular basis for a protein-mediated DNA-bridging mechanism that functions in condensation of the E. coli chromosome
    Pauline Dupaigne
    Centre de Génétique Moléculaire du CNRS, Associé à l Université Paris Sud, 91198 Gif sur Yvette, France
    Mol Cell 48:560-71. 2012
  6. ncbi Role of unusual P loop ejection and autophosphorylation in HipA-mediated persistence and multidrug tolerance
    Maria A Schumacher
    Department of Biochemistry, Duke University School of Medicine, Durham, NC 27710, USA
    Cell Rep 2:518-25. 2012
  7. pmc Structural mechanism of ATP-induced polymerization of the partition factor ParF: implications for DNA segregation
    Maria A Schumacher
    Department of Biochemistry, Duke University School of Medicine, Durham, North Carolina 27710, USA
    J Biol Chem 287:26146-54. 2012
  8. pmc Implications for proteasome nuclear localization revealed by the structure of the nuclear proteasome tether protein Cut8
    Kojiro Takeda
    G0 Cell Unit, Okinawa Institute of Science and Technology, 1919 1 Tancha, Onna, Okinawa, Japan
    Proc Natl Acad Sci U S A 108:16950-5. 2011
  9. pmc Structures of carbon catabolite protein A-(HPr-Ser46-P) bound to diverse catabolite response element sites reveal the basis for high-affinity binding to degenerate DNA operators
    Maria A Schumacher
    Department of Biochemistry and Molecular Biology, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
    Nucleic Acids Res 39:2931-42. 2011
  10. pmc Plasmid protein TubR uses a distinct mode of HTH-DNA binding and recruits the prokaryotic tubulin homolog TubZ to effect DNA partition
    Lisheng Ni
    Department of Biochemistry and Molecular Biology, University of Texas, M D Anderson Cancer Center, Unit 1000, Houston, TX 77030, USA
    Proc Natl Acad Sci U S A 107:11763-8. 2010

Scientific Experts

  • Maria Schumacher
  • Nam K Tonthat
  • Travis Whitfill
  • Weijun Xu
  • Nagababu Chinnam
  • David S Murray
  • Aneta Dobruk-Serkowska
  • Pauline Dupaigne
  • Kojiro Takeda
  • Mareen Sprehe
  • Lisheng Ni
  • Thomas D Dunham
  • Barbara E Funnell
  • Anthony G Vecchiarelli
  • Sara L Milam
  • Susan H Fisher
  • William Margolin
  • Nam Ky Tonthat
  • Lewis V Wray
  • Irene Ng
  • Marisa Caccamo
  • Kerstyn Bryce
  • Finbarr Hayes
  • Olivier Espeli
  • Fernando Rodríguez-Castañeda
  • Meiyi Wu
  • Frédéric Boccard
  • Daniela Barillà
  • Eugene V Koonin
  • Tiffany Glover
  • Mitsuhiro Yanagida
  • Muthiah Kumaraswami
  • Sarah M McEvoy
  • Laurie K Read
  • John C Fisk

Detail Information

Publications16

  1. pmc Structures of the Bacillus subtilis glutamine synthetase dodecamer reveal large intersubunit catalytic conformational changes linked to a unique feedback inhibition mechanism
    David S Murray
    From the Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239
    J Biol Chem 288:35801-11. 2013
    ..This GSI-α-specific regulatory network could be exploited for inhibitor design against Gram-positive pathogens. ..
  2. pmc The structure of irisin reveals a novel intersubunit β-sheet fibronectin type III (FNIII) dimer: implications for receptor activation
    Maria A Schumacher
    From the Departments of Biochemistry and
    J Biol Chem 288:33738-44. 2013
    ..This finding suggests a possible mechanism for receptor activation by the irisin domain as a preformed myokine dimer ligand or as a paracrine or autocrine dimerization module on FNDC5-like receptors. ..
  3. pmc SlmA forms a higher-order structure on DNA that inhibits cytokinetic Z-ring formation over the nucleoid
    Nam K Tonthat
    Department of Biochemistry, Duke University School of Medicine, Durham, NC 27710, USA
    Proc Natl Acad Sci U S A 110:10586-91. 2013
    ....
  4. pmc Uncoupling of nucleotide hydrolysis and polymerization in the ParA protein superfamily disrupts DNA segregation dynamics
    Aneta Dobruk-Serkowska
    University of Manchester, Manchester M1 7DN, United Kingdom
    J Biol Chem 287:42545-53. 2012
    ..Thus, the architecture of the pocket not only is crucial for optimal ATPase kinetics but also plays a key role in the polymerization dynamics of ParA proteins that drive DNA segregation ubiquitously in procaryotes...
  5. ncbi Molecular basis for a protein-mediated DNA-bridging mechanism that functions in condensation of the E. coli chromosome
    Pauline Dupaigne
    Centre de Génétique Moléculaire du CNRS, Associé à l Université Paris Sud, 91198 Gif sur Yvette, France
    Mol Cell 48:560-71. 2012
    ..Thus, these data reveal the molecular basis for a protein-mediated DNA-bridging mechanism that mediates condensation of a large chromosomal domain in enterobacteria...
  6. ncbi Role of unusual P loop ejection and autophosphorylation in HipA-mediated persistence and multidrug tolerance
    Maria A Schumacher
    Department of Biochemistry, Duke University School of Medicine, Durham, NC 27710, USA
    Cell Rep 2:518-25. 2012
    ....
  7. pmc Structural mechanism of ATP-induced polymerization of the partition factor ParF: implications for DNA segregation
    Maria A Schumacher
    Department of Biochemistry, Duke University School of Medicine, Durham, North Carolina 27710, USA
    J Biol Chem 287:26146-54. 2012
    ..Thus, these data provide insight into a unique mechanism by which a Walker box protein forms polymers that involves the generation of ATP-induced dimer-of-dimer building blocks...
  8. pmc Implications for proteasome nuclear localization revealed by the structure of the nuclear proteasome tether protein Cut8
    Kojiro Takeda
    G0 Cell Unit, Okinawa Institute of Science and Technology, 1919 1 Tancha, Onna, Okinawa, Japan
    Proc Natl Acad Sci U S A 108:16950-5. 2011
    ..The importance of Cut8 for cell viability and its absence in humans suggests it as a possible target for the development of specific chemotherapeutics against invasive fungal infections...
  9. pmc Structures of carbon catabolite protein A-(HPr-Ser46-P) bound to diverse catabolite response element sites reveal the basis for high-affinity binding to degenerate DNA operators
    Maria A Schumacher
    Department of Biochemistry and Molecular Biology, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
    Nucleic Acids Res 39:2931-42. 2011
    ..Indeed, biochemical data show that CcpA-(HPr-Ser46-P) binds the three cre sites with similar affinities. Thus, the data reveal properties that license this protein to function as a global transcription regulator...
  10. pmc Plasmid protein TubR uses a distinct mode of HTH-DNA binding and recruits the prokaryotic tubulin homolog TubZ to effect DNA partition
    Lisheng Ni
    Department of Biochemistry and Molecular Biology, University of Texas, M D Anderson Cancer Center, Unit 1000, Houston, TX 77030, USA
    Proc Natl Acad Sci U S A 107:11763-8. 2010
    ..Finally, we show that the exposed TubZ C-terminal region interacts with TubR-DNA, linking the TubR-bound pBtoxis to TubZ polymerization. The combined data suggest a mechanism for TubZ-polymer powered plasmid movement...
  11. pmc Structure of the Trypanosoma brucei p22 protein, a cytochrome oxidase subunit II-specific RNA-editing accessory factor
    Mareen Sprehe
    Department of Biochemistry and Molecular Biology, University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA
    J Biol Chem 285:18899-908. 2010
    ..Thus, these combined studies suggest that p22 mediates its role in k-RNA editing by acting as an adaptor protein...
  12. pmc Insight into F plasmid DNA segregation revealed by structures of SopB and SopB-DNA complexes
    Maria A Schumacher
    Department of Biochemistry and Molecular Biology, University of Texas, M D Anderson Cancer Center, Unit 1000, Houston, TX 77030, USA
    Nucleic Acids Res 38:4514-26. 2010
    ..This DNA-linking function suggests a novel mechanism for in trans DNA spreading by SopB, explaining how it might mask DNA to prevent DNA-mediated inhibition of SopA polymerization...
  13. pmc Structural basis for ADP-mediated transcriptional regulation by P1 and P7 ParA
    Thomas D Dunham
    Department of Biochemistry and Molecular Biology, MD Anderson Cancer Center, University of Texas, Houston, TX 77030, USA
    EMBO J 28:1792-802. 2009
    ..First, it locks in one specific dimer conformation, and second, it induces the folding of two DNA-binding basic motifs that we show are critical for operator binding...
  14. pmc Molecular mechanisms of HipA-mediated multidrug tolerance and its neutralization by HipB
    Maria A Schumacher
    Department of Biochemistry and Molecular Biology, University of Texas, M D Anderson Cancer Center, Unit 1000, Houston, TX 77030, USA
    Science 323:396-401. 2009
    ..Dimeric HipB interacts with two HipA molecules to inhibit its kinase activity through sequestration and conformational inactivation. Combined, these studies suggest mechanisms for HipA-mediated persistence and its neutralization by HipB...
  15. ncbi P1 partition complex assembly involves several modes of protein-DNA recognition
    Anthony G Vecchiarelli
    Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada
    J Biol Chem 282:10944-52. 2007
    ..This arrangement supports a model in which parS motifs are available for interas well as intramolecular parS recognition...
  16. ncbi Structure of a four-way bridged ParB-DNA complex provides insight into P1 segrosome assembly
    Maria A Schumacher
    Department of Biochemistry and Molecular Biology, University of Texas M D Anderson Cancer Center, Houston, Texas 77030, USA
    J Biol Chem 282:10456-64. 2007
    ..This multibridging capability of ParB is likely critical in its partition complex formation and pairing functions...