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 the dsDNA 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 coli K-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.