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

, David A. Murison

, Ioulia Rouzina

, Penny J. Beuning

, Mark C.

Williams

Department of Physics, Northeastern University, Boston, MA, USA,

Department of Chemistry

and Chemical Biology, Northeastern University, Boston, MA, USA,

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.