Research Abstract:
Our research focuses on obtaining a molecular understanding of the factors that affect the stability and specificity of protein-nucleic acid complexes, as well as their mechanisms of interaction. We are investigating the mechanisms by which duplex DNA is unwound catalytically by a class of motor proteins, called helicases. The unwinding of duplex DNA to form single stranded (ss) DNA is a prerequisite for replication, recombination and repair and this process is catalyzed by DNA helicases, which are ss DNA-stimulated ATPases. These enzymes are ubiquitous and central to all aspects of DNA metabolism. We are investigating several helicases, including E. coli Rep, E. coli UvrD (also known as Helicase II) and E. coli RecBCD, which are involved in DNA replication, repair, and recombination, respectively. Our goal is to understand the mechanism of helicase-catalyzed DNA unwinding and protein translocation along DNA and the role of ATP in these processes. We use thermodynamic, pre-steady state transient kinetic (fluorescence stopped-flow and chemical quenched-flow), single molecule fluorescence (TIRF), structural and molecular biological approaches to investigate these problems.
We are also investigating two single stranded DNA binding proteins, the E. coli SSB protein and the eukaryotic heterotrimeric RPA protein, which bind selectively to ss-DNA and facilitate DNA unwinding by the helicases. The binding of SSB to ss DNA is quite complex, since it binds to ss DNA in a number of different modes which may function selectively in replication, recombination and repair. Using fluorescence and isothermal titration calorimetry, we are investigating the molecular interactions that stabilize the SSB-ss-DNA complexes, the factors that influence the distribution of binding modes as well as the different cooperative behavior in each mode. |
Selected Publications:
Tomko E, Fischer CJ, Niedziela-Majka AM and Lohman TM. A Non-uniform stepping Mechanism for UvrD Monomer Translocation along Single Stranded DNA. Molecular Cell 2004 26: 335-347.
Roy R, Kozlov AG, Lohman TM and Ha T. Dynamic Structural Rearrangements Between DNA Binding Modes of E. coli SSB Protein. J. Molecular Biology 2007 369: 1244-1257.
Lohman TM, Tomko EJ and Wu C. Non-hexameric DNA Helicases and Translocases: Mechanisms and Regulation. Nature Reviews Molecular Cell Biology 2008 9: 391-401.
Wong CJ and Lohman TM. Kinetic Control of Mg2+-dependent Melting of Duplex DNA Ends by E. coli RecBC Helicase. J. Molecular Biology 2008 378: 759-775.
Wu CG and Lohman TM. Influence of DNA End Structure on the Mechanism of Initiation of DNA Unwinding by the E. coli RecBCD and RecBC Helicases. J. Mol. Biol. 2008 382: 312-326. |