Studies concerning the molecular interactions of proteins that bind to nucleic acids are being conducted to elucidate the structure-function relationships that occur during DNA metabolic processes (e.g. synthesis, repair, transcription). We are especially interested in how multiple protein complexes move along nucleic acids and recognize the particular DNA structures at which enzymatic activity occurs.

In particular, we are interested in the regulation of DNA metabolic processes by DNA-dependent ATPases. These enzymes require DNA for ATP hydrolysis, which subsequently leads to alterations in DNA metabolic function. We have identified, purified, cloned, and overexpressed a DNA-dependent ATPase from eukaryotic tissues. Our studies have many phases, which include: enzymology of the ATPase, molecular biological manipulation of the coding sequence, regulation of enzyme activity, exploration of the role of theenzyme in the cell, and the development of novel inhibitors of enzymatic activity. We are using the inhibitors of DNA-dependent ATP hydrolysis to disrupt DNA metabolic processes, and are developing novel chemotherapeutic strategies for treatment of cancer and parasitic diseases.

Additional studies are aimed at elucidating structure-function relationships that occur during DNA metabolic processes. As an example, DNA replication requires that the replication protein complexes move along the DNA template on a millisecond time scale. Consequently, we have developed a method of examining protein-nucleic acid interactions on a sub-millisecond time scale using a Nd-YAG laser, which produces a 5 nanosecond pulse of ultraviolet light. Irradiation of a protein-nucleic acid complex produces a covalent bond between protein and nucleic acid, effectively freezing the protein-nucleic acid interaction for further evaluation by other methods.

We use the laser cross-linking methodology in a number of different systems to ask a variety of questions about the geometry of a DNA-protein complex. For example: what is the geometry of such a complex? How does the geometry change in static versus synthesis modes? How does a replication complex find a 3'-hydroxyl primer-template junction? What is the mechanism of translocation along a DNA template? These questions lead to others that deal with the energy requirements for protein movement and how proteins find each other and their substrates.

Selected References

Hockensmith JW, Kubasek WL, Vorachek WR, von Hippel PH. (1993) "Laser cross-linking of proteins to nucleic acids. I. Examining physical parameters of protein-nucleic acid complexes." J Biol Chem. Jul 268(21):15712-20. [PubMed]

Hockensmith JW, Kubasek WL, Evertsz EM, Mesner LD, von Hippel PH. (1993) "Laser cross-linking of proteins to nucleic acids. II. Interactions of the bacteriophage T4 DNA replication polymerase accessory proteins complex with DNA." J Biol Chem. Jul 268(21):15721-30. [PubMed]

Mesner LD, Truman PA, Hockensmith JW. (1993) "DNA-dependent adenosinetriphosphatase A: immunoaffinity purification and characterization of immunological reagents." Biochemistry. Aug 32(30):7772-8. [PubMed]

Muthuswami R, Mesner LD, Wang D, Hill DA, Imbalzano AN, Hockensmith JW. (2000) "Phosphoaminoglycosides inhibit SWI2/SNF2 family DNA-dependent molecular motor domains." Biochemistry. Apr 39(15):4358-65. [PubMed]

Muthuswami R, Truman PA, Mesner LD, Hockensmith JW. (2000) "A eukaryotic SWI2/SNF2 domain, an exquisite detector of double-stranded to single-stranded DNA transition elements." J Biol Chem. Mar 275(11):7648-55. [PubMed]