Helicase promotes replication re-initiation from an RNA transcript

Abstract

To ensure accurate DNA replication, a replisome must effectively overcome numerous obstacles on its DNA substrate. After encountering an obstacle, a progressing replisome often aborts DNA synthesis but continues to unwind. However, little is known about how DNA synthesis is resumed downstream of an obstacle. Here, we examine the consequences of a non-replicating replisome collision with a co-directional RNA polymerase (RNAP). Using single-molecule and ensemble methods, we find that T7 helicase interacts strongly with a non-replicating T7 DNA polymerase (DNAP) at a replication fork. As the helicase advances, the associated DNAP also moves forward. The presence of the DNAP increases both helicase's processivity and unwinding rate. We show that such a DNAP, together with its helicase, is indeed able to actively disrupt a stalled transcription elongation complex, and then initiates replication using the RNA transcript as a primer. These observations exhibit T7 helicase's novel role in replication re-initiation.

Fig. 1

Non-replicating DNAP prevents helicase slippage and increases unwinding rates. a Cartoon illustrating T7 helicase unwinding and slippage behavior. The helicase unwinds, loses grip, slips, re-grips, and resumes unwinding. Arrows indicate the directions of helicase movements. b, c Representative traces showing the number of unwound base pairs by T7 helicase vs. time in the absence (28 traces in total) or presence (25 traces in total) of non-replicating DNAP, respectively. Experiments were conducted with 2 mM ATP under 8 pN force. For clarity, traces have been arbitrarily shifted along both axes. d Measured processivity of T7 helicase (mean distance between slippage events) in the absence and presence of gp5 and/or trx with 2 mM ATP under 8 pN external force. e T7 helicase unwinding rates between slips as a function of force in the absence or presence of the DNAP with 2 mM ATP. Unwinding rates were determined between slippage events (if any). Error bars represent standard deviations

Fig. 2

Non-replicating DNAP localizes to the fork with the helicase. a A cartoon illustrating helicase-DNAP coupling. A non-replicating DNAP directly interacts with an unwinding helicase. The helicase–DNAP complex is juxtaposed crossed the fork junction. b, c Representative traces showing the force vs. number of base pairs unzipped/unwound in presence of helicase and 2 mM dTTP, either without or with the presence of non-replicating DNAP, respectively. The red curves correspond to unzipping naked DNA. The green arrow indicates a force peak above the naked DNA baseline

Fig. 3

Single-molecule experiments on helicase and non-replicating DNAP collision with a TEC. a Distributions of DNA length increase rates of helicase unwinding with or without replication in 0.5 mM dNTP (each) under 5 pN of force. b Experimental design. Left panel shows a cartoon of E. coli TEC that was stalled at +20 nt from the promoter while a helicase with a non-replicating DNAP encountered the TEC co-directionally. c A representative trace of unwinding without replication after collision. Experiments were carried out in the presence of helicase, DNAP, and 0.5 mM dNTP (each) under 5 pN of force. The dotted line indicates the expected stalled TEC position. The cartoon on the top illustrates replication status after the collision with RNAP. d A representative trace of unwinding with replication after collision. Same experimental conditions were used as in c. For clarity, traces have been shifted along the time axis. e Distributions of DNA length increase rates before (upper panel) and after (lower panel) the collision with RNAP

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

Fig. 5

Proposed T7 replication re-initiation model. Cartoons illustrate a proposed model for T7 replication re-initiation. The replisome here consists of leading-strand DNAP and helicase. When the replisome encounters a leading-strand lesion, helicase may continue to unwind processively via association with a non-replicating DNAP. The two proteins form a complex clamping crossed the fork junction, poising the DNAP for replication re-initiation. After collision with a TEC, the helicase-DNAP displaces the RNAP and the DNAP then uses the RNA as a primer to re-initiate the replication