Engineering Approaches to Biomolecular Motors

Engineering Approaches to Biomolecular Motors: From in vitro to in vivo Poster Abstracts

16-POS Board 16 Single-molecule Characterization of E. coli Pol III Core Polymerase Activity M. Nabuan Naufer 1 , David A. Murison 2 , Ioulia Rouzina 3 , Penny J. Beuning 2 , Mark C. Williams 1 . 1 Department of Physics, Northeastern University, Boston, MA, USA, 2 Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, USA, 3 Department of Chemistry and Biochemistry, Ohio State University, Columbus, OH, USA. Antibiotic resistance is a growing health problem. Hence new antibiotics and new antibiotic targets are critically needed. Bacterial DNA polymerases are potentially attractive targets for the development of new antibiotics because, while the process of DNA replication is conserved, the structures and interactions of the proteins involved vary considerably between prokaryotes and eukaryotes. DNA polymerase III (Pol III) is the replicative DNA polymerase in E. coli and is composed of 10 subunits that tightly coordinate leading and lagging strand synthesis. The core of the polymerase (Pol III core) contains the catalytic polymerase subunit, α, the 3′ → 5′ proofreading exonuclease, ε, and a subunit of unknown function, θ. Here we employ optical tweezers to characterize Pol III core activity on a single DNA molecule. We quantify the transitions between single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) during Pol III core activity via constant force measurements, in which the change in extension can be used to determine the rate of polymerization or exonucleolysis at forces below the melting transition force. These experiments are performed at forces greater than the force at which dsDNA and ssDNA stretching curves cross; therefore, while polymerization is inhibited, exonucleolysis is favored by force. We show that the application of tension facilitates the inter- molecular transfer of the primer between the polymerase and exonuclease subunits of the Pol III core assembly. Here we report the dependence of these catalytic rates on template tension and find the zero-force polymerization rate of Pol III core to be 38 ± 9 nts/s. We show that the dwell times of processive events between pauses for polymerization and exonucleolysis are 0.7 ± 0.1 s and 0.3 ± 0.1 s, respectively, suggesting distinct DNA binding dynamics during the polymerase and exonuclease activities.

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