Research Topics
Department of Microbiology
Ph.D. (1975) Marquette University
Research Topics
Research Interests
The research in my laboratory is centered around the genetics and biochemistry of DNA binding proteins. Three of the proteins we are studying are required for site-specific recombination by bacteriophage l. One phage-encoded protein, Integrase (Int), participates in the formation of a higher-order protein-DNA complex called the intasome and performs the strand exchanges that form recombinant products. We have isolated mutants that produce Int proteins that are defective in performing distinct steps in the recombination pathway such as intasome formation, synapsis, and strand exchange. The mutant proteins are currently being used to carry out biochemical analyses of the recombination reaction and to construct a structure-function map of Int protein. We are also using a genetic approach to isolate Int mutants that bind altered recognition sites to determine which amino acid residues interact with DNA.
A second phage-encoded protein, Xis, is required for excisive recombination. It participates in the formation of the excisive intasome by bending DNA and by binding cooperatively with Int to form the active recombinogenic complex. We have used a genetic approach to isolate xis mutants that are defective for excisive recombination in vivo. Our results have shown that the amino-terminal domain of Xis is required for DNA binding and that the carboxyl terminal domain is required for cooperative interactions with Int. Future studies will include studies on the mechanism of cooperativity between Xis and Int.
Integration Host Factor (IHF) is a host-encoded protein that is required for both integrative and excisive recombination. In addition, it participates in a variety of cellular processes such as gene expression and regulation, plasmid replication, and the movement of some transposable elements. IHF is a heterodimer that bends DNA when it binds. We have used a genetic approach to isolate IHF binding-site mutants and have used the information to determine which base-pairs in the recognition site are important for recognition by the protein. We have also used a genetic selection to isolate suppressor mutants in the genes that encode the IHF sub-units. The suppressors recognize variant IHF binding sites and the amino acids substituted are believed to be the ones that contact the DNA. We are currently using a biochemical approach towards characterizing the binding properties of the suppressor proteins. A future project involves X-Ray crystal structure analysis of suppressor IHF-DNA complexes
Another project (in collaboration with Dr. A. Salyers, Department of Microbiology) is centered around site-specific recombination of conjugative transposons (CTns). Ctns are large self-transmissible elements that excise from the host chromosome by site-specific recombination to form a circular intermediate. The circular intermediate is nicked at the transfer origin and a single stranded copy is transferred to the recipient cell. A double stranded circular intermediate is produced and integrated into the recipient's genome. Ctns are important because they often carry genes that make cells resistant to antibiotics. Current projects include determining target sites in recipient DNA, defining sites in CTn DNA that bind integrase and host factors, determining the identity of host factors, and constructing in vitro recombination reactions to study the mechanism of recombination.
Two other proteins we study (in collaboration with Dr. R. Gumport, Department of Biochemistry) are the EcoRI restriction endonuclease and methylase enzymes. We are attempting to isolate mutants of the endonuclease that recognize variant sites to create new restriction enzyme specificities. We have also isolated mutants of the methylase that bind DNA normally but do not methylate. DNA sequence analyses of the mutants will determine which amino acid residues are important for catalytic activity.
Key Words
Mechanism of site-specific recombination by bacteriophages and conjugative transposons; Mechanisms of protein recognition of DNA
Current Research Funding NIH