Talk abstracts

Talk on Wednesday 09:15-09:30am submitted by Brian Maxwell

Single-Molecule Investigation of Substrate Binding Kinetics and Protein Conformational Dynamics of a B-Family Replicative DNA Polymerase

Brian Maxwell (Chemistry and Biochemistry, OSU), Zucai Suo (Chemistry and Biochemistry, OSU)

Abstract:
Replicative DNA polymerases use a complex, multistep mechanism for efficient and accurate DNA replication as uncovered by intense kinetic and structural studies. Recently, single-molecule fluorescence spectroscopy has provided new insights into real-time conformational dynamics utilized by DNA polymerases during substrate binding and nucleotide incorporation. We have employed single-molecule Förster Resonance Energy Transfer techniques to investigate the kinetics and conformational dynamics of Sulfolobus solfataricus DNA Polymerase B1 during DNA and nucleotide binding. Our results show that this replicative polymerase can bind to DNA in at least three conformations and can transition between these conformations without dissociating from a primer-template DNA substrate. Furthermore, the relative frequency of each conformation can be affected by the presence of both an incoming nucleotide and any mismatches at the primer-template terminus. Additionally, our analysis has revealed new details of the dissociation kinetics of the polymerase•DNA binary complex. These findings provide novel mechanistic insights into protein conformational dynamics and substrate binding kinetics of a high-fidelity B-family DNA polymerase.

Keywords: Single-molecule, TIR-FRET, DNA Polymerase