Poster abstracts

Poster number 51 submitted by Brian Maxwell

Single Molecule Investigation of the DNA Binding Dynamics of a Replicative DNA Polymerase

Brian Maxwell (Biophysics Program, Ohio State University), Zucai Suo (Biochemistry, Ohio State University)

Abstract:
DNA polymerases are known to undergo a variety of conformational changes during DNA binding, DNA synthesis and proofreading. While they have been well characterized through biochemical techniques and structural studies, single molecule fluorescence spectroscopy can provide new insights into the real time conformational dynamics utilized during DNA binding and the catalysis of nucleotide incorporation. We have employed single-molecule FRET techniques to investigate the kinetics and conformational dynamics of Sulfolobus solfataricus DNA Polymerase B1 (PolB1) during DNA and nucleotide binding. Our results show that PolB1 can bind DNA in at least 2 conformations in the absence of nucleotide, corresponding to the binding of the primer/template terminus in the polymerase or exonuclease active sites. Interestingly we observed that in a small number of binding events, PolB1 is able to transition between the two binding modes without dissociating from the DNA substrates. Furthermore, our results indicate the presence of an additional binding conformation in the presence of correct nucleotides. By analyzing data from many single binding events, we were able to determine the relative fractions of Polymerase binding in each conformation, and calculate the lifetime of each bound state. These findings provide new mechanistic insights into the details of DNA polymerase substrate binding and catalysis.

Keywords: Single Molecule, FRET, DNA Polymerase