Journal: PLoS ONE

Article Title: Utilization of a Deoxynucleoside Diphosphate Substrate by HIV Reverse Transcriptase

doi: 10.1371/journal.pone.0002074

Figure Lengend Snippet: A. 5′ end-labeled primer, annealed to a template strand, was extended in the presence of a range of dADP concentrations by HIV-1 RT. The primer is extended by a single nucleotide, corresponding to the template ‘T’ at the +1 position. Extension reactions contained 10 nM wild-type enzyme, 5 nM primer-template, and were carried out for 10 minutes. Unextended primer is labeled ‘p’, the single nucleotide extension product is ‘p+1’. B. Single nucleotide extension assays of HIV-1 RT or KF exo- (50 nM each) with 10 µM of either dADP or dATP demonstrated no activity in the reaction containing KF exo- and dADP. Lane B, unextended primer. C. Single nucleotide extension assays utilizing dADP, dCDP, dGDP and dTDP incorporation opposite the complementary template nucleotide. Reactions were carried out with 100 µM of the appropriate dNDP or 0.2 µM dNTP, 50 nM enzyme and 5 nM template-primer. Lanes B1 and B2 contained RT and KF exo- respectively, with no nucleotide added. D. The RT preparation does not contain a contaminating nucleotide kinase activity. Nucleotides, which had been pre-incubated in the presence (labeled RT+ADP) or absence of HIV-1 RT, were used in a single nucleotide extension assay with either KF exo- or HIV-1 RT. Lane B: pre-incubation reaction contained no nucleotide. Unextended primer and primer extended by a single nucleotide are indicated ‘p’ and ‘p+1’ respectively.

Article Snippet: Nucleotides, oligonucleotides and commercial enzymes dADP, dCDP, dGDP, dTDP (dNDPs) and dAMP were purchased from MP Biomedicals.

Techniques: Labeling, Activity Assay, Incubation