Talk abstracts

Talk on Thursday 04:45-05:00pm submitted by Brian Maxwell

Global Conformational dynamics of a model Y-family DNA polymerase

Brian A. Maxwell (Biophysics Program, Department of Biochemistry, Ohio State University), Cuiling Xu (Department of Biochemistry, The Ohio State University), Jessica A. Brown (Department of Biochemistry, The Ohio State University), Zucai Suo (Department of Biochemistry, The Ohio State University)

Abstract:
Dpo4 is a member of the Y-family DNA polymerases from the hyperthermophilic organism Sulfolobus solfataricus. Y-family polymerases are characterized by low processivity and selectivity, and are able to recognize and bypass different classes of DNA lesions. A variety of studies have established a minimal reaction pathway common to all DNA polymerases, and it has been proposed that for many polymerases, a conformational change before incorporation of an incoming nucleotide is likely to be the rate-limiting step. However, for Y-family polymerases crystal structure studies show no obvious change following dNTP binding, while single-turnover kinetic assays indicate a rate-limiting conformational change before the dNTP incorporation. Crystal structures have, on the other hand, provided indirect evidence of a translocation event upon dNTP binding. By monitoring Fluorescence Resonance Energy Transfer (FRET) between a donor fluorophore on a DNA substrate and multiple labeled sites in each polymerase domain, we present here a real time picture of the global dynamic conformational changes of Dpo4 during binding and incorporation of a single correct nucleotide. We have found direct evidence for a proposed DNA translocation event followed by a rapid conformational change prior to incorporation and a subsequent slow reverse conformational change after incorporation. Our results show conclusively that a large precatalytic conformational change can not be the rate limiting step in the reaction pathway. Furthermore, we have provided information on the rates and 3D directional and rotational nature of the motions of each domain that characterize the enzyme’s conformational changes during one cycle of catalysis.

References:
1. Fiala KA, Sherrer SM, Brown JA, Suo Z (2008) Mechanistic consequences of temperature on DNA polymerization catalyzed by a Y-family DNA polymerase. Nucleic Acids Res 36: 1990-2001.
2.Wong J. H, Fiala K. A, Suo Z, Ling H (2008) Snapshots of a Y-family DNA polymerase in replication: substrate-induced conformational transitions and implications for fidelity of Dpo4. J Mol Biol 379: 317–330.
3. Xu C, Maxwell BA, Brown JA, Zhang L, Suo Z, 2009 Global Conformational Dynamics of a Y-Family DNA Polymerase during Catalysis. PLoS Biol 7(10): e1000225. doi:10.1371/journal.pbio.1000225

Keywords: DNA polymerase, conformational dynamics, stopped flow FRET