Kenan C Murphy PhD
|Institution||University of Massachusetts Medical School|
|Department||Microbiology and Physiological Systems|
|Address||University of Massachusetts Medical School|
55 Lake Avenue North
Worcester MA 01655
|Institution||UMMS - Graduate School of Biomedical Sciences|
|Department||Molecular Genetics and Microbiology|
|Institution||UMMS - Programs, Centers and Institutes|
|Department||Bacterial Genetics and Pathogenesis|
Ph. D. (1983) University of Maryland
Double-stranded DNA break repair in Escherichia coli
Recombineering technology for gene replacement in bacterial pathogens
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.
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.
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.
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.
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.
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