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Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase

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Science  29 Jan 2010:
Vol. 327, Issue 5965, pp. 590-592
DOI: 10.1126/science.1179595

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.

In vivo studies suggest that replication forks are arrested by head-on transcription complexes, but are unaffected by codirectional transcription complexes (1) [supporting online material (SOM) Text S1]. Mechanisms that resolve head-on collisions in favor of the replisome are therefore necessary for chromosome duplication and may preserve genomic integrity by preventing fork collapse. In vivo data indicate that head-on replisome–RNA polymerase (RNAP) collisions cause chromosomal deletions, which suggests dissociation of the replisome (2). Genetic studies implicate recombinational repair in resolving conflicts between replication and transcription, which also suggests the possibility of fork collapse (3, 4). Similarly, in vitro data imply that the replisome dissociates after encountering a lac repressor, which arrests the fork (5). In contrast, several in vivo studies indicate that although replication forks stall at protein barriers, the replisome remains stable and resumes elongation after removal of the block (6). Thus, replisome stalling may not necessitate fork collapse (7). We investigated the stability of the Escherichia coli replisome after it encounters a head-on RNAP in vitro.

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The E. coli replisome is a multiprotein complex that copies DNA with high speed [~630 nucleotides (nt) s−1] and processivity (~50 kb) (8). A solid-phase assay was used to study a replisome-RNAP head-on collision (Fig. 1A). An RNAP-halted elongation complex was assembled on linear DNA, immobilized to streptavidin beads, then washed with a high concentration of salt to remove unstable RNAP-DNA complexes (fig. S1) (9). Next, the replisome was assembled in two steps: First, the replicative DnaB helicase that encircles the lagging strand was added; second, DNA polymerase III (Pol III), the clamp loader, and the β clamp were added along with ATP (adenosine 5′-triphosphate), [32P]-α-dCTP (2′-deoxycytidine 5′-triphosphate), and [32P]-α-dGTP (2′-deoxyguanosine 5′-triphosphate). Fork movement was initiated by adding [32P]-α-dATP and [32P]-α-dTTP with single-strand DNA binding protein (SSB). We observed a 2.5-kb product equal to the distance from the fork to the promoter, indicating that the replisome was impeded by the RNAP (Fig. 1B, lane 2). Full-length DNA (3.6 kb) was also produced, suggesting incomplete promoter occupancy by RNAP or replisome read-through of the transcription complex (Fig. 1B, lane 2). Omitting the promoter specificity factor, σ70, resulted in only full-length DNA (Fig. 1B, lane 1). An average of 50% (n = 8) of the replisomes produced full-length DNA in the presence of a head-on RNAP (Fig. 1B, right), which exceeded the number of templates that lacked RNAP (24%; fig. S2). This suggested that ~26% of the replisomes passed a head-on RNAP during the 10-min time course. We determined whether the replisome reads-through RNAP directly by performing a pulse-chase experiment in which cold dNTPs (deoxynucleoside triphosphates) were added after 5 min, and extension of the 2.5-kb product was monitored (Fig. 1C). A steady increase in the ratio of full-length to intermediate-length product was observed, indicating that the replisome passes the RNAP, albeit after pausing for a considerable duration (Fig. 1C). The relatively large amount of full-length product (39%) observed after 5 min suggests that some replisomes might have passed the RNAP with high efficiency. Replisome read-through of RNAP on a different template ruled out any sequence-specific effects (fig. S3). The stalled replisome remained active for 60 min after the collision without the need for primosomal proteins, such as PriA/C, that are necessary to reload DnaB onto SSB-coated single-strand DNA (Fig. 1C) (10, 11). DnaB therefore stayed bound to the stalled fork. Consistent with previous studies, we demonstrated that DnaB was required for replication and that SSB prevented DnaB loading in the absence of primosomal proteins (fig. S4). Thus, although the replication fork stalls upon encountering a head-on RNAP, the replisome remains intact and resumes elongation, presumably after displacing the transcription complex.

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. (Right) The mean fraction (± SE) of full-length DNA (0.5, n = 8) produced in the presence of a head-on RNAP is plotted. (C) Time course of leading-strand synthesis on DNA containing a head-on RNAP. The ratio of full-length to intermediate-length DNA (IFL/II) is shown in the right plot (IFL, intensity of full-length DNA; II, intensity of intermediate-length DNA).

We determined whether the replisome displaces the RNAP from DNA by Xho I digestion of the promoter-proximal sequence, which is protected by RNAP (Fig. 2A). RNAP occupancy of the promoter blocks digestion, whereas RNAP displacement allows digestion. Removing ribonucleotides (NTPs) by washing prevents reassembly of the halted RNAP. A significant increase in digestion was observed only when replication was performed (Fig. 2A, right; compare blue and red bars). Little RNAP displacement was detected at 20 min, which is likely due to replication of only 8.5% of the DNA, probably as a result of RNAP binding to the primer-template (12) (fig. S5). Nevertheless, the large increase in digestion due to replication indicates that the replisome displaces the RNAP. Similar results were observed in the presence of GreB and limiting NTPs, which inhibit RNAP backtracking (fig. S6). We further investigated RNAP displacement by monitoring transcription of a challenge template in the presence of σ70 and limiting NTPs. Transcription of the challenge template was only observed after replication, which indicates that the replisome displaced the RNAP (Fig. 2B, compare lanes 2 and 3). These results are consistent with the ability of DnaB to displace a protein block during DNA unwinding (13).

Fig. 2

The replisome displaces a head-on RNAP from DNA. (A) Replisome displacement of RNAP was probed by Xho I digestion of the promoter-proximal sequence in the presence or absence of replication. A halted RNAP was assembled on DNA, and the reaction was divided. Replication was (lanes 2, 4, and 6; blue bars) or was not (lanes 1, 3, and 5; red bars) initiated, and samples were treated with Xho I 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).

Genetic data implicate RNAP modulators such as Mfd in resolving conflicts between replication and transcription (3). Mfd displaces a halted RNAP from DNA and recruits the nucleotide excision repair machinery to the site, which results in preferential repair of the transcribed strand when RNAP stalls at lesion—referred to as transcription-coupled repair (TCR) (1418). TCR has been postulated to promote fork progression by displacing RNAP blocks from DNA (16). We examined whether Mfd facilitates restart of the fork after a head-on collision. After providing 5 min to allow the replisome to collide with RNAP, we divided the reaction and treated it with either Mfd or buffer for a further 5 min. Nearly full extension of the 2.5-kb product was observed upon addition of Mfd (Fig. 3A, compare lanes 1 and 2), which removed the RNAP block (fig. S7). We determined whether Mfd-mediated replication restart requires a supply of DnaB in solution (Fig. 3B). For this experiment, the beads were washed after the collision then resuspended in buffer containing dNTPs and all the replication proteins except DnaB either in the presence or absence of Mfd. The addition of Mfd again resulted in extension of the 2.5-kb product, providing further evidence that DnaB stays bound to the stalled fork. Next, the experiment of Fig. 3A was repeated with Mfd K634N, which is defective in ATP hydrolysis and can no longer dislodge RNAP (17). Mfd K634N failed to promote extension of the 2.5-kb product (Fig. 3C, compare lanes 1 and 2). These results indicate that Mfd promotes fork progression after the collision by using the energy of ATP, and hence translocase activity, to dissociate the transcription complex ahead of the stalled fork. Finally, we demonstrated that the ability of Mfd to promote replication past a head-on RNAP was unaffected by the addition of all four NTPs and GreB, which promote transcription elongation (fig. S8).

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 before (B) or after (A and 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) and (B), lane 2] or K634N Mfd [(C), lane 2] was then added for a further 5 min.

In conclusion, we find that although the replication fork stalls upon collision with a head-on transcription complex, the replisome remains stable and resumes elongation after displacing the RNAP (fig. S9, left). It is conceivable that the collision may induce RNAP backtracking. However, the lack of stimulation of RNAP endonuclease activity after the collision suggests that this is not the case (fig. S10). Moreover, the addition of GreB and NTPs, which inhibit backtracking, have no effect on our assays (figs. S6, S8, and S11). A previous study of the T4 replisome reported that a head-on RNAP remains bound to the DNA, but the RNAP and transcript switch strands as the replication fork passes (19). This result is difficult to reconcile with the current view of transcription. We find that Mfd promotes direct restart of the fork following the collision by facilitating displacement of the RNAP (fig. S9, right). Codirectional collisions are resolved without auxiliary factors; the replisome uses mRNA as a primer to reinitiate leading-strand synthesis after displacing a codirectional RNAP from DNA (9). Pol III extension of the RNA was not observed in our study, probably due to displacement of the transcript. Genetic data suggest that recombinational repair and other RNAP modulators help resolve replisome-RNAP conflicts, thus explaining the normal growth rate of cells lacking Mfd (3). mfd cells, however, demonstrate a greater lapse in replication and cell growth following ultraviolet irradiation, which supports a role for Mfd in facilitating replication through transcription complexes arrested by lesions in vivo (20). Our data demonstrate a new role for TCR in promoting replication past an RNAP block and may have implications for human disorders that result from deficient TCR, such as Cockayne syndrome (15, 16).

Supporting Online Material

www.sciencemag.org/cgi/content/full/327/5965/590/DC1

Materials and Methods

SOM Text

Figs. S1 to S11

References and Notes

  1. We are grateful to S. Darst for providing RNAP and Mfd proteins and expression vectors. We also thank S. Borukhov for providing GreB and D. Zhang for technical support. This work was supported by a grant from the NIH (M.O.D.) and by a Marie-Josee and Henry Kravis Fellowship at the Rockefeller University (R.T.P.).
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