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    Kenan C Murphy PhD

    TitleAssistant Professor
    InstitutionUniversity of Massachusetts Medical School
    DepartmentMicrobiology and Physiological Systems
    AddressUniversity of Massachusetts Medical School
    55 Lake Avenue North
    Worcester MA 01655
    Phone508-856-6042
      Other Positions
      InstitutionUMMS - Graduate School of Biomedical Sciences
      DepartmentMolecular Genetics and Microbiology

      InstitutionUMMS - Programs, Centers and Institutes
      DepartmentBacterial Genetics and Pathogenesis

        Overview 
        Narrative

        Academic Background

        Ph. D. (1983) University of Maryland

        Double-stranded DNA break repair in Escherichia coli
        Recombineering technology for gene replacement in bacterial pathogens

        Photo: Kenan 
Murphy


        My lab is interested in thedsDNA break repair mechanism in Escherichia coli. One mechanism of repair under study is the ssDNA annealing pathway promoted by the bacteriophage lambda Red recombination system. The system consist of two proteins, the ssDNA annealing Bet protein and the 5’-3’ dsDNA lambda exonuclease. These two proteins form a complex in vitro, and are thought to interact with each other in vivo. We have isolated various mutants of Bet that are deficient for recombination, some of which are likely to be incapable of binding to lambda Exo. Our work is designed to analyze the interaction of Bet with Exo to determine if the complex formation is physiologically relevant. For example, does Exo has a role in loading Bet onto single-stranded DNA? Work on this project is aimed at a biochemical description of the interaction between Bet and Exo proteins, and the interaction of the Red complex with dsDNA ends.

        Another dsDNA break repair pathway we are studying is the repair of dsDNA break promoted by the RecN protein. Mutations in the recN gene render E. coli highly susceptible to DNA damaging agents that cause dsDNA breaks (X-rays, crosslinking agents). RecN is an interesting protein. It is greatly induced by SOS, the DNA damage sensing and response program of E. coli. RecN, by sequence comparisons, possesses structural features reminiscent of the SMC (structural maintenance of chromosomes) class of proteins from eukaryotes. Its actual role in DNA repair is unknown. We have constructed plasmids that can be used as substrates for detection of RecN-promoted repair events in vivo. Work on this project involves the genetics and biochemistry of RecN with DNA ends, and the interaction of RecN with other replication and repair functions of E. coli.

        A third avenue of research involves the use of Red recombineering technology for gene replacement in bacterial pathogens. My lab was the first to show that the lambda Red recombination system promotes gene replacement of electroporated linear DNA substrates into the Escherichia coliK-12 chromosome at a very high efficiency (Murphy, 1998). We have shown that the system is also useful in pathogenic species of E. coli, where in one experiment, five pathogenicity islands of enterohemorrhagic E. coli(EHEC) were easily deleted and replaced with a kanamycin marker (Murphy & Campellone, 2003). Work continues to adapt the Red recombineering technology to other bacterial pathogens such as Pseudomonas aeruginosa and Mycobacterium tuberculosis.

        replication Kenan Murphy figure page

        Proposed mechanism of Red Recombineering

        Lambda Exo's 5' exonuclease activity generates ssDNA, which serves as a substrate for Bet-promoted annealing to ssDNA in the lagging strand of a replication fork. The red line in the recombinant could represent a gene deletion, a small insertion or point mutation.



        Rotation Projects

        Rotation Projects

        1. Study the interaction of the ssDNA annealing protein Bet and its cognate exonuclease with dsDNA ends. Compare wild type Bet with one or mutant Bet proteins. Project involves a protein isolation, protein-DNA binding assays, and basic molecular biological techniques.

        2. Electroporate linearized plasmids into E. coli wild type and recN mutants and follow the repair of the dsDNA ends by the emergence of drug resistant colonies. Determine the genetic requirements for efficient RecN-promoted repair. Project involves electroporation experiments, basic bacterial plating experiments and molecular biological techniques.

        3. Help develop recombineering technology for Pseudomonas aeruginosa by the electroporation of PCR substrates into electrocompetent cells. Project involves electroporation experiments, basic bacterial plating experiments and molecular biological techniques.


        Bibliographic 
        selected publications
        List All   |   Timeline
        1. Murphy KC, Volkert MR. Structural/functional analysis of the human OXR1 protein: identification of exon 8 as the anti-oxidant encoding function. BMC Mol Biol. 2012; 13(1):26.
          View in: PubMed
        2. Murphy KC. Phage recombinases and their applications. Adv Virus Res. 2012; 83:367-414.
          View in: PubMed
        3. Flockhart AF, Tree JJ, Xu X, Karpiyevich M, McAteer SP, Rosenblum R, Shaw DJ, Low CJ, Best A, Gannon V, Laing C, Murphy KC, Leong JM, Schneiders T, La Ragione R, Gally DL. Identification of a novel prophage regulator in Escherichia coli controlling the expression of type III secretion. Mol Microbiol. 2012 Jan; 83(1):208-23.
          View in: PubMed
        4. Brady MJ, Radhakrishnan P, Liu H, Magoun L, Murphy KC, Mukherjee J, Donohue-Rolfe A, Tzipori S, Leong JM. Enhanced Actin Pedestal Formation by Enterohemorrhagic Escherichia coli O157:H7 Adapted to the Mammalian Host. Front Microbiol. 2011; 2:226.
          View in: PubMed
        5. Tree JJ, Roe AJ, Flockhart A, McAteer SP, Xu X, Shaw D, Mahajan A, Beatson SA, Best A, Lotz S, Woodward MJ, La Ragione R, Murphy KC, Leong JM, Gally DL. Transcriptional regulators of the GAD acid stress island are carried by effector protein-encoding prophages and indirectly control type III secretion in enterohemorrhagic Escherichia coli O157:H7. Mol Microbiol. 2011 Jun; 80(5):1349-65.
          View in: PubMed
        6. Murphy KC. Targeted chromosomal gene knockout using PCR fragments. Methods Mol Biol. 2011; 765:27-42.
          View in: PubMed
        7. Murphy KC, Ritchie JM, Waldor MK, Løbner-Olesen A, Marinus MG. Dam methyltransferase is required for stable lysogeny of the Shiga toxin (Stx2)-encoding bacteriophage 933W of enterohemorrhagic Escherichia coli O157:H7. J Bacteriol. 2008 Jan; 190(1):438-41.
          View in: PubMed
        8. Murphy KC. The lambda Gam protein inhibits RecBCD binding to dsDNA ends. J Mol Biol. 2007 Aug 3; 371(1):19-24.
          View in: PubMed
        9. Campellone KG, Roe AJ, Løbner-Olesen A, Murphy KC, Magoun L, Brady MJ, Donohue-Rolfe A, Tzipori S, Gally DL, Leong JM, Marinus MG. Increased adherence and actin pedestal formation by dam-deficient enterohaemorrhagic Escherichia coli O157:H7. Mol Microbiol. 2007 Mar; 63(5):1468-81.
          View in: PubMed
        10. Savage PJ, Leong JM, Murphy KC. Rapid allelic exchange in enterohemorrhagic Escherichia coli (EHEC) and other E. coli using lambda red recombination. Curr Protoc Microbiol. 2006 Jan; Chapter 5:Unit5A.2.
          View in: PubMed
        11. Murphy KC, Campellone KG. Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli. BMC Mol Biol. 2003 Dec 13; 4:11.
          View in: PubMed
        12. Murphy KC, Campellone KG, Poteete AR. PCR-mediated gene replacement in Escherichia coli. Gene. 2000 Apr 4; 246(1-2):321-30.
          View in: PubMed
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