Ortiz Theodore P, Marshall Jason A, Meyer Lauren A, Davis Ryan W, Macosko Jed C, Hatch Jeremy, Keller David J, Brozik James A
Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131, USA.
J Phys Chem B. 2005 Aug 25;109(33):16127-31. doi: 10.1021/jp051778f.
DNA polymerases are protein machines that processively incorporate complimentary nucleotides into a growing double-stranded DNA (ds-DNA). Single-base nucleotide incorporation rates have been determined by stalling and restarting various polymerases, but intrinsic processive rates have been difficult to obtain, particularly for polymerases with low processivity, such as the human immunodeficiency virus type 1 reverse transcriptase (HIV RT) polymerase. Here we find, using a new fluorescence-based single-molecule polymerization assay, that the intrinsic processive DNA-dependent polymerization of HIV RT is approximately Poissionian (i.e. each nucleotide is added sequentially) with a rate of about 100 bases per second at 21 degrees C. From the same experiments, based on the stepping statistics of polymerization, we also estimate the rates for HIV RT early termination and final release of completely replicated primer-template DNA. In addition, by measuring the rate of polymerization as a function of temperature, we have estimated the activation energy for processive nucleotide incorporation.
