Fig. 5. Pre-steady-state kinetics of α-borano-d4T monophosphate and d4T monophosphate incorporation into DNA as described (Kati et al., 1992). (A) Pre-steady-state kinetics of a single α-borano-d4T monophosphate addition. A 5′-³²P-labelled 21mer was annealed to a 31mer DNA template (100 nM) specifying a single thymidine insertion site immediately adjacent to the 3′ end of the primer, and reverse transcriptase (30 nM active sites) was allowed to bind (Canard et al., 1998). The reaction was initiated by the addition of 100 µM α-borano-d4T triphosphate, 6 mM MgCl₂ in a rapid quench apparatus (Kin Tek, State college, PA) at 37°C, and quenched at various times with 0.3 M EDTA. Products were analysed by denaturing gel electrophoresis and quantitated using photostimulatable plates and a Fuji Imager. Data were fitted to the burst equation: product = A(1 – e^–kt) + k_sst, where A is the amplitude, k is the apparent pre-steady-state rate and k_ss is the steady-state rate. (B) Partial data set of pre-steady-state rates determined as described above using 2.5 (filled circles), 5 (crosses), 10 (open circles), 20 (open triangles) and 60 µM (filled squares) α-borano-d4T triphosphate, 6 mM MgCl₂. (C) First-order rates determined as described above of α-borano-d4T triphosphate and d4T triphosphate plotted against nucleotide analogue concentration to determine K_D (µM) and k_pol (per second) using a hyperbolic fit of the data (Kati et al., 1992).