Single molecule study of DNA polymerization by HIV-1 reverse transcriptase

PHYS 477

Sangjin Kim,, Charles Schroeder,, Paul C. Blainey,, and X. Sunney Xie, Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford street, Cambridge, MA 02138
We use a flow-stretched DNA assay to investigate the primer extension activity of HIV-1 reverse transcriptase (RT) at the single molecule level. The assay consists of multi-functionalized single-stranded lambda-phage DNA molecules stretched by hydrodynamic force. DNA molecules serve as individual templates for wild-type HIV-1 RT. In this manner, we observe plus-strand DNA synthesis of reverse transcription in an in vitro setting. To directly observe slow enzymatic activity, through-objective dark field microscopy is implemented to image micron-sized beads tethered to DNA molecules. We measure polymerization rate and processivity of the enzyme as a function of enzyme concentration, temperature, and template stretching force. Furthermore, we study the enzymatic rate and location of dissociation events as a function of base-pair sequence, and we discuss the force-dependent enzymatic behavior for its implication to the chaperoning role of nucleocapsid proteins during reverse transcription in vivo.

Poster Session
7:30 PM-10:00 PM, Wednesday, 13 September 2006 Moscone Center -- Hall D, Poster

8:00 PM-10:00 PM, Monday, 11 September 2006 Moscone Center -- Hall D, Sci-Mix

Division of Physical Chemistry

The 232nd ACS National Meeting, San Francisco, CA, September 10-14, 2006