Direct restart of a replication fork stalled by a head-on RNA polymerase

Abstract

In vivo studies suggest that replication forks are arrested by encounters with head-on transcription complexes. Yet, the fate of the replisome and RNA polymerase (RNAP) after a head-on collision is unknown. We found that the Escherichia coli replisome stalls upon collision with a head-on transcription complex, but instead of collapsing, the replication fork remains highly stable and eventually resumes elongation after displacing the RNAP from DNA. We also found that the transcription-repair coupling factor Mfd promotes direct restart of the fork after the collision by facilitating displacement of the RNAP. These findings demonstrate the intrinsic stability of the replication apparatus and a previously unknown role for the transcription-coupled repair pathway in promoting replication past a RNAP block.

Fig. 1. The replisome slowly passes a head-on RNAP

(A) A head-on RNAP was assembled on immobilized DNA. DnaB was added, then the replisome was assembled and replication initiated. Replisome component functions (8): Pol III core (orange), synthesizes DNA; β-clamp (dark blue), confers processivity to Pol III; clamp-loader (light blue), assembles β-clamps onto primed sites; DnaB (yellow), unwinds DNA. (B) Leading strand synthesis was performed in the presence (lane 2) or absence (lane 1) of a head-on RNAP. The mean fraction of full-length DNA (0.5, n=8) produced in the presence of a head-on RNAP is plotted with standard error (right). (C) Time course of leading strand synthesis on DNA containing a head-on RNAP. The ratio of full-length to intermediate-length DNA (I_FL/I_I) is shown in the right plot (I_FL = intensity of full-length DNA, I_I = intensity of intermediate-length DNA).

Fig. 2. The replisome displaces a head-on RNAP from DNA

(A) Replisome displacement of RNAP was probed by XhoI digestion of the promoter-proximal sequence either in the presence or absence of replication. A halted RNAP was assembled on DNA and the reaction was divided. Replication was (lanes 2, 4, 6, blue bars) or was not (lanes 1, 3, 5, red bars) initiated and aliquots were treated with XhoI at the indicated times. RU = relative units. (B) RNAP displacement in the presence (lane 3) or absence (lane 2) of replication was determined by monitoring transcription of a challenge template. Transcription of challenge template control (lane 1).

Fig. 3. Mfd promotes fork progression following a head-on collision

Leading strand synthesis was performed on DNA containing a head-on RNAP. Reactions were divided prior to (B) or after (A,C) replication was performed for 5 min. (B) DnaB was removed from solution by washing after the collision was formed. Buffer (lanes 1) or either wild-type (A,B, lane 2) or K634N Mfd (C, lane 2) was then added for a further 5 min.