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

PHYS 477

Sangjin Kim, kim7@fas.harvard.edu, Charles Schroeder, cschroed@fas.harvard.edu, Paul C. Blainey, blainey@fas.harvard.edu, and X. Sunney Xie, xie@chemistry.harvard.edu. 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

Sci-Mix
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