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The following HTML text is provided to enhance online readability. Many aspects of typography translate only awkwardly to HTML. Please use the page image as the authoritative form to ensure accuracy. DNA helicase (15, 16), that is responsible for catalyzing nascent strand regression at stalled forks. In this study, the presence of transcribing RNA polymerase on UV-irradiated chromosomes was shown to promote replication-fork stalling and lethality in ∆ruvAC mutant strains. Survival of UV-irradiated ∆ruv strains was found to be enhanced in derivatives carrying either spoT1 [which increases the steady-state level of the RNA polymerase modulator (p)ppGpp] or rpo* [mutations in rpoB that mimic the effect of (p)ppGpp binding to RNA polymerase]. This ruv-independent recovery relied heavily on UvrABC-mediated excision repair and on the activities of PriA, RecG, and RecA. The need for PriA implied that replication forks had stalled and that the replication machinery had to be reassembled at sites remote from oriC. RecBCD was not required, and the typical UV sensitivity of recB mutations could only be observed in the presence of the Holliday junction resolvase RusA. These observations led us to propose that RecG-catalyzed replication fork regression occurred at forks stalled at UV lesions in the template and that replication restart proceeded via a pathway that did not involve D loop formation by RecBCD-mediated recombination. This finding was supported by the demonstration that RecG, but not RuvAB, could convert three-way junctions to Holliday junctions that could be cleaved by a resolvase such as RusA or RuvC. Here we have examined the fate of nascent DNA formed in vitro on plasmid templates carrying oriC where replication forks have been stalled as a result of the absence of a topoisomerase. These early θ-type replication intermediates could be cleaved by either RusA or RuvC. RusA cleavage was inhibited by RuvA and, in intermediates where the replisome had been inactivated, by negative supercoiling, suggesting that Holliday junctions had indeed formed spontaneously in the positively supercoiled early replication intermediate (ERI). RecG stimulated RusA cleavage of ERIs where the replisome had been inactivated and promoted cleavage when the inactivated ERI was negatively supercoiled. These results suggest that under normal conditions in the cell, when the replicating chromosome is expected to be maintained at a net negative linking difference, the action of RecG, or enzymes with similar activities, catalyzes nascent strand regression at stalled replication forks. Materials and MethodsReplication and Recombination Proteins. Proteins for oriC replication—DnaA, DnaB, DnaC, DnaG, HU, the single-stranded DNA-binding protein (SSB), the DNA polymerase III holoenzyme (Pol III HE) (reconstituted from preparations of Pol III* and the β subunit), topoisomerase IV (Topo IV), and DNA gyrase—were purified as described (17, 18). RusA (19), RuvA and RuvB (20), RuvC (21), and RecG (22) were purified as indicated. oriC DNA Replication. Standard oriC DNA replication reaction mixtures (20 µl) were as described by Hiasa and Marians (17) except that no topoisomerase was added so that ERI accumulated. Template DNA was plasmid pBROTB535 type I, a 6-kb-long molecule (23). Gel electrophoretic analyses of replication products were performed as described (17). ResultsHolliday Junctions Form in Early Replication Intermediates Containing Paused Replication Forks. We used the oriC DNA replication system to generate paused replication forks that could then be probed to detect Holliday junction formation. We took advantage of the fact that the plasmid DNA templates used in the replication system are negatively supercoiled. Thus, initiation can occur in the absence of a topoisomerase. Replication fork progression then occurs until excess positive windings accumulate, typically as positive supercoils. We have demonstrated that this intermediate (the ERI) is a true kinetic intermediate in the replication pathway (22). In the oriC replication system, the ERI contains a nascent leading strand that is about 1 kb in length (Fig. 2). Although at first glance this seems longer than expected, we have shown recently that under these conditions, only one of the two forks that are formed at oriC is released to form the ERI (N. Smelkova and K.J.M., unpublished data). And, as shown here, origin-proximal regression of the nascent DNA in the ERI allows more fork progression than one would have predicted based on the superhelical density of the starting template DNA. Further replication fork progression in the ERI requires relief of the accumulated topological strain by either adding a topoisomerase or cutting one of the template strands (24). It recently has been demonstrated, by using intermediates made with the oriC replication system (25), that the ERI, in fact, migrates during electrophoresis through neutral agarose gels as if it were relaxed, rather than containing supercoils of any type. This finding suggested that the positive supercoiling generated during replication actually promoted nascent strand regression. This result is because the preferred resting state of closed circular DNA is the relaxed form, negatively supercoiled molecules thus tend to favor unwinding of the duplex turns, whereas positively supercoiled molecules favor rewinding of the duplex turns. Either process results in the removal of supercoils from the respective molecule. Thus, in a positively supercoiled ERI, the tendency to rewind the template strands actually drives nascent strand regression. Postow et al. (25) were able to observe the reversed fork in purified replication intermediates by using scanning force microscopy. We wanted to examine whether fork regression was occurring during the replication reaction, thus the use of analytical techniques such as scanning force microscopy were not applicable. Instead, we reasoned that if nascent strand regression occurred in the ERI, the Holliday junction formed should be susceptible to cleavage by the Holliday junction-specific resolvases RusA and RuvC. Cleavage of ERIs containing Holliday junctions can generate three major species (Fig. 1). Presumably, either the origin-proximal, origin-distal, or both ends of the nascent DNA can pair to generate four-way junctions. Cleavage at only one of these points generates an a structure (Fig. 1iii). This DNA form will migrate slightly slower than the ERI in neutral agarose gels. If both ends of the nascent DNA regress and both Holliday junctions in the same molecule are cut, two products will be generated, distinguished by the orientation of resolution. In one case, a nicked circular (form II) molecule and a small duplex fragment corresponding to the distance along the nascent DNA between the resolution points will arise (Fig. 1v). In the other case, a nicked linear DNA will arise that is longer by the remaining regions of nascent DNA than the linear form (form III) of the original plasmid template (Fig. 1vi). This DNA form (labeled as form III′ in the figures) will migrate with a mobility that is intermediate between that of form II and form III DNAs on neutral agarose gels. To examine whether Holliday junctions formed in ERIs where replication forks had paused because of accumulated topological strain, RusA and RuvC were included in oriC replication reactions that contained DnaA, DnaB, DnaC, DnaG, HU, the single-stranded DNA-binding protein (SSB), and the DNA polymerase III holoenzyme (Pol III HE), but no topoisomerase. Analysis of the products of reaction by neutral agarose gel electrophoresis revealed that, indeed, the ERI could be cleaved by the Holliday junction resolvases, generating at least a structures and form II DNA (Fig. 2A). Cleavage could be detected at lower concentrations of RusA than RuvC. As expected from previous studies (26), cleavage of the parental strands removed the topological constraint and thus released the paused replication fork. This release allowed extension of the nascent leading strand to full length (Fig. 2B) and suggested that, under these
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