Studies of the structure-function relationship of protein-protein and protein-DNA interactions related to DNA repair
We use atomic force microscopy to study association constants and conformational properties of protein-protein and protein-DNA complexes. In collaboration with Tom Kunkel and Sam Wilson at NIEHS, we are interested in studying two main DNA repair systems: DNA mismatch repair and DNA base excision repair.
DNA mismatch repair
DNA mismatch repair (MMR) is a highly conserved repair pathway essential to the maintenance of genomic stability. This repair mechanism corrects errors post-replicatively, including mismatches in base-pairing and small insertion or deletion loops. The exact mechanism through which proteins repair mismatched DNA, however, is not fully understood. We use AFM to study the structural and functional properties of protein-DNA complexes essential to the initiation of DNA mismatch repair.
DNA base excision repair
The replication of DNA is known to occur with high fidelity; however, errors are often made in the replication process that must be repaired to maintain genomic stability. Base excision repair (BER) is a major repair mechanism that focuses specifically on repairing aberrant bases and filling base gaps. There are many proteins essential to BER, and these generally form large complexes necessary for repair to occur. We are studying the oligomerization states of some of these repair proteins as well as the binding affinities of these proteins to each other and to nicked DNA. We are also interested in DNA structural changes induced by BER proteins, which will provide further insight into their biological function.
Development of a combined AFM-fluorescence microscope for the study of multi-protein systems
AFM is a powerful technique for studying protein-protein and protein-DNA interactions, however it is often difficult to distinguish between two or more unique proteins in multi-protein complexes using AFM alone. We are developing a microscope that combines atomic force microscopy with fluorescence to enable us to study multi-protein systems while distinguishing between unique proteins in those complexes. By combining the forces of AFM and fluorescence resonance energy transfer (FRET), we are able to determine relative positions and stoichiometries of multi-protein systems as well as visualize small proteins that are invisible to the AFM. This microscope will be used to the study multi-protein interactions essential to DNA repair.
Single-molecule fluorescence spectroscopy of protein-DNA complexes
AFM results have revealed different DNA-protein conformations at a base mismatch in the initiation of DNA mismatch repair. The dynamics that exist between these two conformations, however, cannot be acquired with AFM alone. We are developing a single-molecule fluorescence system (in conjunction with Nancy Thompson's lab) to further explore the key features of mismatch recognition and to gain kinetic information about the recognition steps of DNA repair. We use single-pair fluorescence resonance energy transfer (spFRET) to analyze conformational changes in DNA as mismatch repair proceeds. These experiments will elucidate, in great detail, the structural and functional dynamics of DNA mismatch repair, and open the door for the subsequent (and more complex) stages of DNA mismatch repair to be studied.
Transient-state kinetic studies of single and multiple nucleotide incorporation
We are using rapid kinetic methods in an effort to elucidate the mechanism of RNA synthesis catalyzed by E. coli RNA polymerase. Experiments include characterization by single nucleotide incorporation. In conjunction, we are examining the rate of double nucleotide incorporation using the wild type DNA template as well as mutants of the template DNA strand to better understand downstream sequence affects on the rate of nucleotide incorporation. We are also studying the mechanism of transcription elongation via the phenomenon of misincorporation, or the addition of the incorrect base to the growing RNA chain. The data from these experiments is being used to further develop the proposed mechanism of transcription.
Finally, to understand the regulation of transcription, we are now investigating how accessory proteins affect the kinetics using T. thermophilus RNA polymerase. Such studies should reveal how elongation accessory proteins regulate synthesis.
Characterization of RNA polymerases from thermophilic bacteria
A large number of studies indicate that E. coli RNA polymerase as well as several eukaryotic RNA polymerases undergo multiple conformational changes during transcription. To investigate the role of conformational transitions in the regulation of transcription, we have begun a comparative study of E. coli and T. thermophilus RNA polymerases. Comparison of different species of bacterial RNA polymerases should provide insight into the role of conformational transitions in the regulation of transcription.
Following the recent publication of the structures of T. thermophilus and T. aquaticus RNA polymerase, we are currently investigating the roles of individual residues in the protein. Utilizing site directed mutagenesis to change key residues in the structure, we can further our understanding of the exact behavior of the RNA polymerase during transcription.