Fig. 4.

Bulk experiments on helicase and non-replicating DNAP collision with a TEC. a, b Experiments on fork DNA substrate (left) and blunt DNA substrate (right) respectively. The parental DNA contained a stalled T7 RNAP and experiments were carried out with dNTP mixture spiked with [α-³²P]-dGTP. For each experiment, reactions were quenched at four time points (0, 60, 180, and 600 s). Samples were mixed with formamide and bromophenol blue dye and heated at 95 °C for 5 min before loading on 12% acrylamide/6M urea sequencing gels. Sequencing gels show the kinetics of the RNA primer extension on either the fork substrate or the blunt substrate. The run-off DNA product is 38-nt long. The products running close to the 10-nt DNA markers are 14-mer and 15-mer resulting from dNTPs addition by T7 RNAP to the 12-mer RNA primer. c Replication reaction performed with just the primer annealed to the template. Experiment was carried out with dNTP mixture spiked with [α-³²P]-dGTP and T7 DNAP. Reaction was quenched at 600 s. The replication product was used as a control for quantitating the % run-off DNA products obtained in a and b. d Percentage run-off product estimated from a and b