Recombination and fork progression in bacteriophage T4

Summary

Principal Investigator: Stephen W White
Abstract: DESCRIPTION (provided by applicant): Homologous recombination is a fundamental event in DNA metabolism. Long recognized for its role in generating genetic diversity, recombination is now known to be crucial for DNA repair and the rescue of stalled replication forks. Defects in these repair mechanisms in higher organisms lead to the accumulation of mutations that eventually result in cancer, and the proposed studies are therefore directly relevant to human disease. We are interested in understanding the underlying mechanisms of recombination at the structural level, and propose to study them in a very simple, well characterized organism, namely bacteriophage T4. T4 is an ideal system for these studies because it relies on recombination-dependent replication or RDR and efficient replication fork progression to generate the required levels of DNA during its infection cycle in Escherichia coli. Seven T4 proteins will be studied, UvsX, UvsY, UvsW, UvsW.1, Dda, gp32 and endonuclease VII. The recombination protein triad UvsX, UvsY and UvsW mediate the core of the homologous recombination reaction and are related to the eukaryotic proteins Rad51, Rad52 and Rad54, respectively. UvsW and Dda are helicases that translocate and/or unwind branched nucleic acid structures and have important roles in recombination and replication fork progression. Defects in helicases such as Bloom and Werner are known to cause cancer in humans, and there is evidence that UvsW and Dda may function very similarly to these molecules. UvsW.1 is a previously unknown T4 protein that we have identified, with a putative role in recombination. gp32 is the T4 single-stranded DNA binding protein that is known to have crucial roles in many aspects of T4 DNA metabolism. Finally, endonuclease VII resolves Holliday Junctions to complete the homologous recombination reaction. The mechanisms of, and interactions between, these seven proteins will be studied at the molecular level by a coordinated approach involving X-ray crystallography to study their structures, in vitro methods to study their individual functions and interactions, and in vivo methods to understand their biological roles. A considerable body of preliminary data has been obtained for this project that includes crystal and NMR structures, important preliminary crystals, purified proteins, demonstrations of biochemical activities, and in vivo function based on analysis of T4 mutants.
Funding Period: 2004-08-01 - 2015-05-31
more information: NIH RePORT

Top Publications

  1. ncbi Mutation of a conserved active site residue converts tyrosyl-DNA phosphodiesterase I into a DNA topoisomerase I-dependent poison
    Xiaoping He
    Department of Structural Biology, St Jude Children s Research Hospital, Memphis, TN 38105, USA
    J Mol Biol 372:1070-81. 2007
  2. pmc The phage T4 protein UvsW drives Holliday junction branch migration
    Michael R Webb
    Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA
    J Biol Chem 282:34401-11. 2007
  3. ncbi Crystallographic and NMR analyses of UvsW and UvsW.1 from bacteriophage T4
    Iain D Kerr
    Department of Structural Biology, St Jude Children s Research Hospital, 332 N Lauderdale Street, Memphis, TN 38105, USA
    J Biol Chem 282:34392-400. 2007
  4. pmc Fork regression is an active helicase-driven pathway in bacteriophage T4
    David T Long
    Department of Biochemistry, Duke University Medical Center, Box 3711, Durham, North Carolina 27710, USA
    EMBO Rep 10:394-9. 2009
  5. pmc Crystal structure of the phage T4 recombinase UvsX and its functional interaction with the T4 SF2 helicase UvsW
    Stefan Gajewski
    Department of Structural Biology, St Jude Children s Research Hospital, Memphis, TN 38105, USA
    J Mol Biol 405:65-76. 2011
  6. pmc The T4 phage SF1B helicase Dda is structurally optimized to perform DNA strand separation
    Xiaoping He
    Department of Structural Biology, St Jude Children s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
    Structure 20:1189-200. 2012
  7. pmc Architecture of the bacteriophage T4 activator MotA/promoter DNA interaction during sigma appropriation
    Meng Lun Hsieh
    From the Gene Expression and Regulation Section, Laboratory of Cell and Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892
    J Biol Chem 288:27607-18. 2013
  8. pmc Coordination and processing of DNA ends during double-strand break repair: the role of the bacteriophage T4 Mre11/Rad50 (MR) complex
    Joshua R Almond
    Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
    Genetics 195:739-55. 2013
  9. pmc DNA damage responses in prokaryotes: regulating gene expression, modulating growth patterns, and manipulating replication forks
    Kenneth N Kreuzer
    Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
    Cold Spring Harb Perspect Biol 5:a012674. 2013

Research Grants

  1. The MRAD9 Radioresistance Gene
    Howard B Lieberman; Fiscal Year: 2013
  2. Northeast Biodefense Center
    W Ian Lipkin; Fiscal Year: 2013
  3. Host-tumor cell interaction in myeloma: therapeutic applications
    Kenneth C Anderson; Fiscal Year: 2013
  4. Cellular Responses to DNA-Protein Crosslinks
    Amanda K McCullough; Fiscal Year: 2013
  5. MECHANISTIC STUDIES OF GENETIC RECOMBINATION
    STEPHEN CHARLES KOWALCZYKOWSKI; Fiscal Year: 2013
  6. Novel Interactions of DNA Repair Processes in Replication Fork Maintenance
    Priscilla K Cooper; Fiscal Year: 2013
  7. Electron Microscopy of Biological Macromolecules
    Kenneth H Downing; Fiscal Year: 2013
  8. THE BIOCHEMISTRY OF GENETIC RECOMBINATION/RECA PROTEIN
    Michael M Cox; Fiscal Year: 2013
  9. Spatial and Temporal Regulation of Angiogenesis
    HAROLD FISHER DVORAK; Fiscal Year: 2013
  10. Roles of the RecQ Helicases BLM and RECQ5 in Genome Maintenance
    Patrick Sung; Fiscal Year: 2013

Detail Information

Publications9

  1. ncbi Mutation of a conserved active site residue converts tyrosyl-DNA phosphodiesterase I into a DNA topoisomerase I-dependent poison
    Xiaoping He
    Department of Structural Biology, St Jude Children s Research Hospital, Memphis, TN 38105, USA
    J Mol Biol 372:1070-81. 2007
    ..However, the distinct pattern of mutant Tdp1 activity evident in yeast cells, suggests a more severe defect in Tdp1H(432)N-catalyzed resolution of 3' phospho-adducts...
  2. pmc The phage T4 protein UvsW drives Holliday junction branch migration
    Michael R Webb
    Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA
    J Biol Chem 282:34401-11. 2007
    ..Taken together, these results strongly support a role for UvsW in the branch migration of Holliday junctions that form during T4 recombination, replication, and repair...
  3. ncbi Crystallographic and NMR analyses of UvsW and UvsW.1 from bacteriophage T4
    Iain D Kerr
    Department of Structural Biology, St Jude Children s Research Hospital, 332 N Lauderdale Street, Memphis, TN 38105, USA
    J Biol Chem 282:34392-400. 2007
    ..The NMR solution structure of UvsW.1 reveals a dynamic four-helix bundle with homology to the structure-specific nucleic acid binding module of RecQ helicases...
  4. pmc Fork regression is an active helicase-driven pathway in bacteriophage T4
    David T Long
    Department of Biochemistry, Duke University Medical Center, Box 3711, Durham, North Carolina 27710, USA
    EMBO Rep 10:394-9. 2009
    ..We also show that UvsW resolves purified fork intermediates in vitro by fork regression. Regression is therefore part of an active, UvsW-driven pathway of fork processing in bacteriophage T4...
  5. pmc Crystal structure of the phage T4 recombinase UvsX and its functional interaction with the T4 SF2 helicase UvsW
    Stefan Gajewski
    Department of Structural Biology, St Jude Children s Research Hospital, Memphis, TN 38105, USA
    J Mol Biol 405:65-76. 2011
    ..Finally, we show that the phage helicase UvsW completes the UvsX-promoted strand-exchange reaction, allowing the generation of a simple nicked circular product rather than complex networks of partially exchanged substrates...
  6. pmc The T4 phage SF1B helicase Dda is structurally optimized to perform DNA strand separation
    Xiaoping He
    Department of Structural Biology, St Jude Children s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
    Structure 20:1189-200. 2012
    ..The conserved interface between the pin and the SH3 domain provides the mechanism for tight coupling of translocation to strand separation...
  7. pmc Architecture of the bacteriophage T4 activator MotA/promoter DNA interaction during sigma appropriation
    Meng Lun Hsieh
    From the Gene Expression and Regulation Section, Laboratory of Cell and Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892
    J Biol Chem 288:27607-18. 2013
    ..Our results demonstrate the utility of fine resolution FeBABE mapping to determine the architecture of protein-DNA complexes that have been recalcitrant to traditional structure analyses. ..
  8. pmc Coordination and processing of DNA ends during double-strand break repair: the role of the bacteriophage T4 Mre11/Rad50 (MR) complex
    Joshua R Almond
    Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
    Genetics 195:739-55. 2013
    ..These results indicate that the nuclease activity of Mre11 is critical for phage growth and recombination-dependent replication during T4 infections. ..
  9. pmc DNA damage responses in prokaryotes: regulating gene expression, modulating growth patterns, and manipulating replication forks
    Kenneth N Kreuzer
    Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
    Cold Spring Harb Perspect Biol 5:a012674. 2013
    ..Finally, some recent advances that relate to the replication and repair of damaged DNA will be summarized. ..

Research Grants31

  1. The MRAD9 Radioresistance Gene
    Howard B Lieberman; Fiscal Year: 2013
    ..abstract_text> ..
  2. Northeast Biodefense Center
    W Ian Lipkin; Fiscal Year: 2013
    ..As a Center based in a School of Public Health and a State Department of Health, the NBC has a firm commitment to and practical understanding of Emergency Preparedness. ..
  3. Host-tumor cell interaction in myeloma: therapeutic applications
    Kenneth C Anderson; Fiscal Year: 2013
    ..Ultimately, our goal is to validate MM-host cell interactions as a target for novel therapeutics to improve patient outcome in MM. ..
  4. Cellular Responses to DNA-Protein Crosslinks
    Amanda K McCullough; Fiscal Year: 2013
    ..Collectively, these investigations will yield comprehensive analyses of repair and tolerance of this class of DNA lesions. ..
  5. MECHANISTIC STUDIES OF GENETIC RECOMBINATION
    STEPHEN CHARLES KOWALCZYKOWSKI; Fiscal Year: 2013
    ..Failure to repair DNA properly can ultimately lead to cancer development, premature aging, and anemia. ..
  6. Novel Interactions of DNA Repair Processes in Replication Fork Maintenance
    Priscilla K Cooper; Fiscal Year: 2013
    ....
  7. Electron Microscopy of Biological Macromolecules
    Kenneth H Downing; Fiscal Year: 2013
    ....
  8. THE BIOCHEMISTRY OF GENETIC RECOMBINATION/RECA PROTEIN
    Michael M Cox; Fiscal Year: 2013
    ..Ref may eventually be utilized in the genomic engineering of mammalian DNA, in the service of human gene therapy or the generation of mouse gene knockouts. ..
  9. Spatial and Temporal Regulation of Angiogenesis
    HAROLD FISHER DVORAK; Fiscal Year: 2013
    ..abstract_text> ..
  10. Roles of the RecQ Helicases BLM and RECQ5 in Genome Maintenance
    Patrick Sung; Fiscal Year: 2013
    ..In this renewal project, we will strive to delineate the multi-faceted role of BLM in HR repair and regulation, and to also define the mechanism underlying the novel role of RECQ5 as an anti-recombinase. ..