Protein–DNA complexes are the primary sources of replication fork pausing in Escherichia coli

RecD2 Inhibits Resumption of Replication by Paused Replisomes.

Superfamily 1 helicases that translocate 3′-5′ along ssDNA (E. coli Rep and UvrD and Bacillus stearothermophilus PcrA) promote movement of E. coli replisomes through nucleoprotein complexes, whereas D. radiodurans RecD2 and bacteriophage T4 Dda, 5′-3′ superfamily 1 helicases do not (10). Indeed, addition of RecD2 results in an apparent increase in the degree of replication blockage at protein–DNA complexes in vitro (10). We investigated the basis for this apparent increase in fork blockage by RecD2. We used a system in which replisomes could be blocked completely by a large array of lac repressor–operator complexes and then the block relieved by subsequent addition of isopropyl β-D-1-thiogalactopyranoside (IPTG), allowing monitoring of the ability of blocked replisomes to resume replication (18)(Fig. 1A and Fig. S1). Replisomes were reconstituted on plasmid templates bearing oriC and 22 tandem lac operators. In the absence of a topoisomerase, replisomes could proceed only a limited distance along the template owing to accumulation of positive supercoiling. Continued fork movement depended on cleavage of the template by a restriction enzyme to relieve the topological strain (17) (Fig. 1A, ii and iii and Fig. S1) with the site of cleavage allowing only one of the two forks to progress toward the lac operators (Fig. 1A, iii and iv).

In the absence of lac repressor, replication generated a population of lagging strands of ∼0.5 kb and leading strands of 1.3 and 5.2 kb (Fig. 1A, vi and B, lane 1). In the presence of repressor, 3.5-kb, rather than 5.2-kb, leading strands were generated (Fig. 1A, vi and B, lane 2 and Fig. S1), as expected if movement of replisomes was inhibited within the repressor–operator array. Subsequent addition of IPTG to these blocked forks relieved this inhibition (Fig. 1B, lane 3), reflecting the initial retention of activity of replisomes blocked at lac repressor–operator complexes (18).

The impact of helicases on the ability of blocked replisomes to resume replication upon addition of IPTG was analyzed. Rep, UvrD, and PcrA did not promote replication through the 22 repressor–operator complexes in the absence of IPTG (Fig. 1B, lanes 5, 7, and 9). Addition of IPTG allowed resumption of replication even in the presence of these helicases, indicating that Rep, UvrD, and PcrA did not inhibit resumption of replication upon removal of a block (Fig. 1 B, i, lanes 4, 6, and 8 and 1 B, ii). In contrast, RecD2 reduced severely the ability of replisomes to resume replication after IPTG addition (Fig. 1 B, i, compare lanes 10 and 11 with lanes 3 and 2, and 1 B, ii). However, a mutant RecD2 protein, pinless RecD2, that retains DNA binding but not helicase activity (23) failed to inhibit resumption of replication (Fig. 1C). The helicase activity of RecD2 thus inhibits continued movement of paused replisomes upon dissociation of a blocking nucleoprotein complex. T4 Dda, another superfamily 1 5′-3′ helicase, also inhibited resumption of fork movement (Fig. S2), demonstrating that fork inactivation is a general feature of this class of helicases rather than an activity associated specifically with RecD2.

RecD2 Inactivates Paused but Not Elongating Replisomes.

Inhibition of resumption of fork movement upon dissociation of the protein–DNA block could be explained by RecD2-catalyzed inactivation of paused replisomes, of elongating replisomes, or both. To distinguish between these possibilities we assessed the impact of RecD2 on the activity of elongating and of paused forks in two parallel experiments. First, the effects of wild-type and pinless RecD2 were analyzed on elongating replication forks whose movement around a supercoiled plasmid template lacking engineered barriers was sustained by DNA gyrase (Fig. 2 A, i). Addition of either wild-type or pinless RecD2 alongside the replication initiator DnaA had no significant impact on levels of DNA synthesis by elongating forks (Fig. 2 A, ii). Second, replication of the same supercoiled plasmid template was initiated by DnaA in the absence of a topoisomerase as in Fig. 1, resulting in fork stalling owing to topological strain (Fig. 2 B, i). Subsequent addition of a restriction enzyme relieved the strain and allowed paused forks that retained function to continue (Fig. S1). However, addition of wild-type, but not pinless, RecD2 alongside the restriction enzyme inhibited subsequent DNA synthesis (Fig. 2 B, ii). RecD2 helicase activity results, therefore, in inactivation of forks paused by positive supercoiling. Taken together, the data in Fig. 2 indicate that RecD2 inactivates paused but not elongating replisomes.

RecD2 Inactivates Forks Paused by a Variety of Replicative Barriers.

Different obstacles have different impacts on replisome movement. Nucleoprotein complexes and topological strain require removal of the original block for resumption of replication (Figs. 1 and 2). In contrast, DNA lesions pause forks but can eventually be bypassed. A cyclobutane pyrimidine dimer within the leading strand template pauses fork progression, but leading strand synthesis can be reinitiated downstream of the lesion over the course of several minutes (11). We tested, therefore, whether RecD2 inhibited the ability of replisomes paused at a pyrimidine dimer to bypass the lesion and continue replication.

Reactions were again initiated in the absence of a topoisomerase followed by restriction enzyme cleavage in the presence of labeled deoxynucleotide to allow forks to progress. The plasmid template contained a single pyrimidine dimer within the leading strand template (11). Following restriction enzyme cleavage for 50 s, excess unlabeled deoxynucleotide was added with or without RecD2 or pinless RecD2 and incubation continued. One minute after addition of the restriction enzyme all reactions contained predominantly stalled replication forks (Fig. 3B, lanes 1, 5, and 9) (11). In the absence of RecD2 the majority of stalled forks generated full-length products after 8 min (Fig. 3B, lanes 1–4). Wild-type, but not pinless, RecD2 inhibited this conversion of stalled forks into full-length products (Fig. 3B, compare lanes 5–8 with 9–12, and 3C). RecD2 helicase therefore prevents replisomes paused by a DNA lesion from bypassing the DNA damage.

The data in Figs. 1–3 indicate that RecD2 helicase activity results in loss of function of paused, but not elongating, replisomes regardless of the nature of the replicative barrier.

Absence of Rep Helicase Hypersensitizes Cells to recD2 Expression.

Inactivation of paused but not elongating replication forks by RecD2 in vitro provides a potential tool to probe fork pausing in vivo. Conversion of paused forks that retain the ability to continue replication into inactivated forks that cannot continue upon removal or bypass of blocks might present viability problems, the severity of which would depend upon the frequency and duration of fork pausing. However, induction of recD2 expression from a plasmid-based arabinose-inducible promoter had no significant impact on viability as monitored by colony-forming ability (10) (Fig. 4 A and B, i). Chromosomal DNA content of wild-type cells induced for recD2 expression was affected significantly, however, as monitored by flow cytometry under run-out conditions (Fig. 4E, i). Thus, RecD2 did have an impact upon chromosome duplication. This inhibition of chromosomal duplication required RecD2 helicase activity, not just DNA binding, because recD2pinless had no impact on DNA content (Fig. 4E, ii). Sensitivity to wild-type but not pinless RecD2 in vivo correlates with inactivation of paused forks by wild-type but not pinless RecD2 in vitro (Figs. 1C, 2B, and 3).

The ability to survive recD2 expression could be due to repair of RecD2-inactivated forks providing an efficient means of surviving such inactivation. Recombination enzymes provide multiple pathways to repair damaged replication forks (24, 25). However, no significant impact on viability was observed in the absence of any recombination gene tested, suggesting that fork processing is not critical for survival of cells expressing recD2 (Fig. 4A; ΔrecA, ΔrecB, and ΔrecF) (Table S1).

Alternatively, survival could reflect a low frequency and/or short duration of replication fork pauses in wild-type cells. Low levels of replisome pausing might be due to an inherently low frequency of pausing but might also be a consequence of multiple enzyme systems in wild-type cells that minimize fork pausing. We therefore analyzed the impact of recD2 expression in strains with a decreased ability to remove potential obstacles to replication. Lesions within the leading strand template cause replisome pausing before bypass of the lesion via repriming (11) (Fig. 3). However, absence of nucleotide excision repair did not render cells sensitive to RecD2 (Fig. 4A, ΔuvrA, ΔuvrB, and ΔuvrC). Absence of Mfd, a dsDNA translocase that promotes transcription-coupled repair of DNA lesions via UvrABC (26), also did not render cells sensitive to RecD2 (Fig. 4A). Thus, removal of bulky lesions from either nontranscribed or transcribed DNA was not required for tolerance of recD2 expression. Similarly, absence of one or both of the apurinic/apyrimidinic endonucleases required for base excision repair did not result in hypersensitivity to RecD2 (Fig. 4A, ΔxthA and Δnfo). Efficient removal of DNA lesions, and thus a reduction in their potential to pause forks, was not required therefore for tolerance of recD2 expression.

Rep helicase provides another means to reduce fork pausing by acting at the replisome to disrupt nucleoprotein complexes ahead of the replication fork (10, 22, 27). Wild-type but not pinless RecD2 had a severe impact on colony-forming ability in the absence of Rep (Fig. 4 A and B). Absence of Rep also caused hypersensitivity to T4 Dda (Fig. S3A). The toxicity of both RecD2 and Dda in Δrep cells correlated, therefore, with the ability of both helicases to inactivate replisomes in vitro. The requirement for Rep to ameliorate the impact of RecD2 on colony-forming ability was dependent on Rep helicase activity, not just DNA binding, because a rep allele bearing a mutation in the Walker A motif was sensitive to RecD2 (Fig. 4C, iii). Thus Rep does not merely block access of RecD2 to a DNA substrate. Chromosomal DNA content upon recD2 induction in Δrep cells was also reproducibly more severely affected compared with rep⁺ cells (Figs. 4E, i and iii and 5E, i and F, i).

Taken together, these data indicate that inactivation of paused but otherwise functional replisomes by RecD2 results in chromosomal defects that, although exhibited by wild-type cells, are exacerbated in the absence of Rep. The corollary of these observations is that many fork pausing events do not lead to replisome inactivation under normal circumstances in either rep⁺ or Δrep cells and that conversion of these paused forks into inactive forks by RecD2 is deleterious.

Clearance of Nucleoprotein Barriers Ahead of Forks Protects Against RecD2-Induced Cell Death.

Rep facilitates both clearance of nucleoprotein complexes ahead of forks (10, 22) and PriC-directed reloading of the replication apparatus (28, 29). However, the colony-forming ability of a strain lacking PriC was not reduced by RecD2 (Fig. 4A) or T4 Dda (Fig. S3B), indicating that failure of PriC-directed replisome reloading is not responsible for the inviability of Δrep cells expressing RecD2.

Efficient clearance of nucleoprotein complexes ahead of forks by Rep is dependent upon not only Rep helicase activity but also a physical interaction between the C terminus of Rep and the replicative helicase DnaB (10, 30). A rep allele lacking the DnaB interaction domain but retaining helicase activity (repΔC33) was sensitive to RecD2 expression (Fig. 4D), consonant with efficient clearance of protein–DNA complexes ahead of forks being needed to minimize RecD2-induced viability defects. Both UvrD and DinG helicases have also been implicated in promoting replication of protein-bound DNA (10, 22), but neither helicase is known to associate physically with the replisome (31). Overexpression of recD2 had no impact on colony-forming ability in the absence of either UvrD or DinG (Fig. 4A), supporting the conclusion that this toxicity is specific for the replisome-associated activity of Rep.

These data indicate that promotion of replisome movement by Rep along protein-coated DNA may be essential to avoid RecD2-directed cell death. A major source of nucleoprotein replicative barriers is transcription, with mutations within RNA polymerase subunits being able to suppress multiple defects in genome duplication (20, 32–34). One such mutation, rpoB[H1244Q], suppresses defects in genome duplication by reducing the stability of stalled transcription complexes (20) and inhibiting backtracking of RNA polymerase (35). We found that this mutation suppressed the RecD2-dependent killing of Δrep cells as evinced by colony-forming ability (Fig. 5 A–C, compare i and ii with iii and iv, and 5D). Flow cytometry was also used to analyze DNA content. Although these analyses used rifampicin, an inhibitor of transcription initiation, to prevent replication reinitiation and simplify DNA content analysis, comparison of rpoB⁺ with rpoB[H1244Q] revealed that rpoB[H1244Q] reduced the impact of RecD2 on chromosomal DNA content in both rep⁺ and Δrep cells (compare i and ii in Fig. 5 E and F). However, suppression by rpoB[H1244Q] in Δrep cells was only partial, with both colony sizes reduced and DNA content still perturbed significantly by high level expression of recD2 (Fig. 5C, iii and iv, and F, ii).

Other mutations in rpo genes with the ability to suppress genome duplication defects also restored colony-forming ability to Δrep cells, confirming that this suppression was not specific to rpoB[H1244Q] (Fig. 5B, v–viii). Indeed, rpoB[G1260D] (33) resulted in effective suppression of RecD2 toxicity even with high-level recD2 expression, as indicated by colony sizes and DNA content in Δrep cells (Fig. 5C, compare v and vi, and 5F, compare i and iii), suppression that was also apparent with dda expression in Δrep cells (Fig. S3C). This suppression of toxicity suggested that transcription elongation factors might also provide a key means of ameliorating the consequences of paused fork inactivation. However, the simultaneous absence of four factors known to reduce RNA polymerase pausing and backtracking (26) did not render cells sensitive to recD2 expression (Fig. S4).

These data indicate that transcription complexes are the primary cause of RecD2-directed DNA content defects in both rep⁺ and Δrep cells and of lethality in Δrep cells. Together with our demonstration that RecD2 inactivates paused forks in vitro, these findings indicate that the most significant cause of paused replisomes in otherwise unperturbed cells in vivo are transcription complexes. Moreover, the primary means of resuming replication from these paused replisomes is via an accessory replicative helicase.

Plasmids, Proteins, and Strains.

Construction of pPM561, pBADrecD2, pBADrecD2pinless, pBADdda, and pMG31 are described in SI Methods.

LacI, Rep, UvrD, β, HU, DnaA, DnaB, DnaC, DnaG, single-stranded binding protein (SSB), and DNA polymerase III α, ε, θ, τ, χ, ψ, δ, and δ′ subunits were purified as described (10, 13, 37, 38). B. stearothermophilus PcrA, D. radiodurans wild-type and pinless RecD2, E. coli DNA gyrase, and T4 Dda were kind gifts of Panos Soultanas (University of Nottingham, Nottingham, UK), Dale Wigley (Chester Beatty Laboratories, Institute of Cancer Research, London, UK), Tony Maxwell (John Innes Centre, Norwich, UK), and Kevin Raney (University of Arkansas for Medical Sciences, Little Rock, AR). EagI was supplied by New England Biolabs. All protein concentrations refer to the monomer.

Strains are listed in Table S2.

In Vitro Replication Assays.

Assays to monitor the functionality of forks blocked by nucleoprotein complexes or positive supercoiling are described in SI Methods. To analyze the impact of test helicases on the ability of replisomes to replicate plasmid DNA in the absence of a replicative block (Fig. 2A), reactions containing pPM561 were assembled containing DNA gyrase (140 nM) but without LacI and replication was initiated as outlined in SI Methods. For analysis of replisome stability at a cyclobutane pyrimidine dimer, proteins and DNA template were prepared as described (11) and reactions performed as described in SI Methods.

Colony Formation Assays and Flow Cytometry.

Strains were transformed with the indicated plasmids and colonies selected on LB agar containing either 100 μg⋅mL⁻¹ ampicillin (pMG31) or 30 μg⋅mL⁻¹ kanamycin (pBADrecD2, pBADrecD2pinless, and pBADdda) at 37 °C overnight. Single colonies were grown subsequently in LB broth plus either ampicillin or kanamycin at 37 °C to an A₆₅₀ of 0.4 before serial 10-fold dilutions were made with 56/2 salts on ice. Five microliters of the dilutions were then spotted onto LB agar containing ampicillin or kanamycin plus the indicated concentrations of arabinose. Plates were incubated at 37 °C for 16 h. Flow cytometry was performed on midlog phase cultures after treatment with rifampicin and cephalexin as described in SI Methods.