INTRODUCTION

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Initiation of DNA replication from bacterial and eukaryotic origins generally involves limited unwinding within an A-T-rich region, promoted by either initiator protein(s) or an origin transcript (for reviews, see references 6, 8, and 16). The unwound region provides an assembly site for the replication complex including the replicative helicase, which catalyzes extensive unwinding of the parental strands. In plasmids containing the ColE1 replicon and in mitochondrial DNA (for reviews, see references 22 and 34), RNA polymerase (RNAP) generates an origin transcript that forms a persistent RNA-DNA hybrid (R-loop). The transcript holds open the origin region for assembly of the replisome and also serves as the primer for leading-strand synthesis.

Regulation of DNA replication is usually exerted at the stage of initiation. In the case of plasmid ColE1, regulation is achieved by expression of an anti-sense RNA (RNA I) that is complementary to the 5' end of the primer RNA (RNA II). RNA I binds to RNA II and prevents it from folding into a conformation favorable for hybridization with the template (for reviews, see references 16 and 22). The detailed mechanism of regulation in both eukaryotic and prokaryotic chromosomal origins is not known, in part due to the complexities of the initiation events and of cell cycle regulation. In eukaryotic systems, a complex network of protein phosphorylation and dephosphorylation is involved in regulation. Although several eukaryotic replication proteins are phosphorylated (i.e., MCM proteins, DNA polymerase alpha-primase), the specific phosphorylation events that regulate initiation remain to be determined (for a review, see reference 37). In both eukaryotic and prokaryotic systems, proteins that bind and hydrolyze ATP are involved in the early steps of DNA replication (Cdc6p and DnaC, respectively), suggesting that ATP binding and hydrolysis may act as a molecular switch (for a review, see reference 25). This step may provide additional control by preventing reinitiation of replication at the origins, thus limiting replication to once per cell cycle.

Bacteriophage T4 uses two major replication initiation mechanisms, which are regulated to occur only during certain phases of the infective cycle (for reviews, see references 21 and 31). At early times of infection, replication proceeds mainly by the origin-dependent mode from any of several replication origins. Expression of phage-encoded recombination proteins results in the onset of recombination-dependent replication, which is the predominant mode during late times of infection. Throughout the phage life cycle, DNA replication is tightly coupled to transcription, recombination, and repair.

Modification of the Escherichia coli RNAP results in the temporal regulation of T4 gene expression (for a review, see reference 30). Immediately after infection, unmodified host RNAP recognizes T4 early promoters and begins the transcription of early genes. The early promoters are turned off and middle-mode promoters are activated upon expression of two phage-encoded proteins, AsiA (binds ⁷⁰) and MotA (binds middle-mode promoters). Finally, upon synthesis of gp33 and gp55, the latter being a replacement  factor (38), transcription is shifted from middle to late promoters.

Two well-characterized T4 origins of replication, ori(uvsY) and ori(34), consist of a middle-mode promoter juxtaposed to a DNA unwinding element (29). These origins have been cloned into a plasmid vector, and both replicate autonomously during T4 infection (17; for a review, see reference 21). Previous in vivo analyses using permanganate sensitivity provided a strong argument that the origin transcript forms an R-loop within the DNA unwinding element of plasmid-borne ori(uvsY) (4). Two-dimensional gel analyses of T4 genomic replication intermediates indicated that the transcript within the R-loop serves as the primer for leading-strand synthesis (1). Consistent with this model, putative RNA-DNA copolymers have been identified within the downstream region of ori(uvsY) (31, 35). Furthermore, a synthetic origin R-loop was recently shown to be an excellent substrate for the T4 in vitro replication system, with the RNA serving as the primer for leading-strand synthesis (31a).

Two models have been proposed for the repression of T4 origin-dependent replication at late times of infection. In the first model, origin-dependent replication is blocked when the host RNAP is converted to the late form, which cannot synthesize the origin transcript (28). The second model proposes that a phage-encoded protein produced late during T4 infection, UvsW, is a repressor of origin function that dissociates the origin R-loop (3, 7).

The earliest evidence that UvsW protein represses origin-dependent replication was that uvsW mutations (including nulls) restored the arrested DNA synthesis caused by mutations that inactivate T4 recombination proteins (5, 40, 41). Experiments with plasmid model systems suggested that the uvsW mutations did not restore the arrested DNA synthesis by directly rescuing recombination-dependent replication but rather by activating an alternative mode of late replication (7). The presence of five helicase motifs (including the Walker A motif) in the amino acid sequence of UvsW provided a strong hint that the protein functions as a helicase. Indeed, UvsW was purified as a glutathione S-transferase (GST) fusion protein and shown to exhibit both DNA-dependent ATPase and DNA helicase activities on branched substrates (3). Furthermore, a point mutation within the Walker A motif (K141R) eliminated both ATPase and helicase activities.

In this study, we demonstrate that one of the functions of the UvsW protein is to dissociate the origin R-loop through an RNA-DNA helicase activity. The dissolution of the R-loop leads to the switch from origin-dependent replication to recombination-dependent replication. Two-dimensional gel analyses show that the replicative intermediates formed at ori(uvsY) persist for a longer time during a uvsW mutant infection than in a wild-type T4 infection. The same method also shows that origin replicative intermediates are abolished when UvsW is produced artificially at early times. Finally, using a synthetic R-loop substrate, we show that purified UvsW efficiently dissociates the RNA from the R-loop.

	MATERIALS AND METHODS

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Materials. Restriction enzymes, Taq DNA polymerase, and T4 DNA ligase were purchased from various commercial sources, radiolabeled nucleotides were from Dupont NEN, and Nytran membranes were from Schleicher and Schuell. Oligonucleotides were synthesized by Sigma-Genosys Biotechnologies, Inc. GST-UvsW fusion proteins were purified as described by Carles-Kinch et al. (3). Luria broth (L broth) contained Bacto tryptone (Difco Laboratories) (10 g/liter), yeast extract (Difco Laboratories) (5 g/liter), and NaCl (10 g/liter).

E. coli and phage strains. E. coli strains included CR63 (K12, supD ^r) (9), BL21 (F ompT hsdS_B [r_B m_B] gal dcm lon), BL21 (DE3) (as BL21 with  DE3), and AB1 [araD139 (ara-leu)7697 lacX74 galU galK hsdR rpsL], as described by Kreuzer et al. (19). Bacteriophage T4 strains included K10 (amB262 [gene 38] amS29 [gene 51] nd28 [denA] rIIPT8 [denB-rII deletion]) (33), K10-116 (as K10, with an 8-bp linker insertion within uvsY that renders the phage uvsY) (19), and K10-uvsW1 (as K10, with uvsW1, a 1.3-kb HindIII deletion within uvsW that renders the phage uvsW) (7).

Plasmids. Plasmid pKK405 is a pBR322 derivative harboring a 1.4-kb HindIII T4 fragment with ori(uvsY) (17, 18). pKCK41 is a pET11d derivative harboring the T4 uvsW gene under the control of a mutated T7 promoter (3). pKCK42 is a control plasmid for pKCK41 with a large fragment of the uvsW gene in the opposite orientation (relative to the promoter) (3). pKCK43 is a derivative of pKCK41 with the uvsW gene containing the K141R point mutation (3). pKCD444 is a derivative of pKCK41 with a ClaI deletion (uvsW2) that removes most of the uvsW gene downstream of the promoter.

Two-dimensional agarose gel analysis. For the experiment shown in Fig. 2, BL21 cells were grown in L broth at 37°C to an optical density at 560 nm (OD₅₆₀) of 0.5 and then infected with K10 or K10-uvsW-K141R at a multiplicity of 6 PFU/cell. At the indicated time points, 1.5-ml aliquots were removed, cell pellets were collected by centrifugation, and total nucleic acids were purified as previously described (19). For the experiments shown in Fig. 4 and 5, BL21 (DE3) cells harboring the indicated plasmid were grown to an OD₅₆₀ of 0.250 in L broth at 37°C, isopropyl--D-thiogalactopyranoside (IPTG) was added (100 µM), and the cells were further incubated at 37°C until the OD₅₆₀ reached 0.5. T4 strain K10 was then added at a multiplicity of infection of 6 PFU/cell and samples were collected as described above.

Northern blot analysis. RNA isolation and Northern blot analyses were performed as described by Belanger and Kreuzer (1). The oligonucleotide probe for the uvsW transcript, 5'-CGAAAATAGCTTTACCATGCG-3', was 5'-end labeled using T4 kinase in the presence of [-³²P]dATP. After analysis of the uvsW transcript, the probe was washed from the blot, and the blot was reprobed with a ³²P-labeled oligonucleotide probe for the ori(uvsY) transcript, 5'-CCCAATACTGCAACGAG-3'.

Transcription of RNA for the synthetic R-loop. The template for in vitro transcription reactions consisted of a PCR product with a mutated T7 promoter juxtaposed to ori(uvsY) DNA [positions 5 to +99 with respect to the ori(uvsY) promoter transcription] (29). The mutations in the T7 promoter were necessary to maintain complete sequence homology between the transcript and ori(uvsY) DNA. The PCRs included linear pKK405 DNA as the template in conjunction with specific primers T75 (5'-GCTTCGAAATTAATACGACTCACTATAGAATCTAAGTCCATCCAT-3') and T7+99 (5'-GCGGTCGCGAATCTATAAACACATCTTTCTTC-3'; BstUI restriction site underlined). The two-stage PCR protocol consisted of the following steps: 94°C, 2 min; 45°C, 1 min; and 50°C, 3 min for 10 cycles in the first stage; and 94°C, 1 min; 60°C, 1 min; and 60°C, 2 min for 30 cycles in the second stage. The PCR products were gel purified and digested with BstUI to generate a blunt-ended template for in vitro transcription reactions (Megashortscript kit; Ambion Company). The 104-base transcript was labeled by incorporation of [-³²P]UTP residues, and the RNA concentration was determined by specific activity based on the nucleotide concentrations in the transcription reaction.

Preformed R-loop at ori(uvsY). The ori(uvsY) synthetic R-loop was generated by a modification of the protocol of Lee and Clayton (26). Briefly, 10 pmol of plasmid DNA was mixed with the indicated amount of ori(uvsY)-specific transcript in 100-µl reaction mixtures in R-loop buffer (62% formamide, 400 mM NaCl, 25 mM HEPES [pH 7.5], 1.25 mM EDTA). The mixture was incubated at 62°C for 4 h; the temperature was slowly reduced (1°C every 8.5 min) to 42°C, held at 42°C for 12 to 16 h, slowly cooled to 37°C (1°C per h), and held at 37°C for 1 h; and the mixture finally was incubated at room temperature for an additional 1 h. Where indicated, free RNA was separated from R-loop on a CL-4B Sepharose column equilibrated with R-loop buffer. R-loop-containing fractions were determined by radioactivity and by agarose gel electrophoresis followed by autoradiography, and fractions were stored at 4°C.

BglII inhibition assay. The R-loop was monitored by inhibition of cleavage at a BglII site located 60 bp downstream of the ori(uvsY) transcription start site. Approximately 0.25 pmol of plasmid or R-loop was incubated at room temperature with 25 U of BglII for 1 h. The reaction was stopped with sodium dodecyl sulfate, and the products were applied to a 0.6% agarose gel (in 0.5× TBE buffer). Following electrophoresis, the DNA was analyzed by Southern blotting with a radioactive probe generated from pKK405 plasmid DNA using the Random Primed kit (Roche Molecular Biochemicals). Bands were quantitated using an AMBIS 100 Imager (AMBIS Inc.).

Mapping the RNA within the R-loop. The R-loop transcript was mapped within pKK405 by digestion with HindIII, which cuts the plasmid into two fragments. The R-loop (0.12 pmol) was first treated with 1 M glyoxal at 14°C for 2 h (14, 24) to modify the displaced strand, precipitated at 80°C for 2 h, and then incubated with HindIII (100 U) for 35 min at 37°C. The digests were analyzed by agarose gel electrophoresis followed by autoradiography.

R-loop unwinding assay. Formamide was removed from the R-loop by chromatography through a 1-ml Sephadex G-50 spin column. The unwinding reaction mixtures (20 µl) contained approximately 80 fmol of R-loop substrate in 20 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 2 mM dithiothreitol, 5 mM ATP (or as indicated in the Fig. 7 legend), bovine serum albumin at 100 µg/ml, and the indicated amounts of either purified GST-UvsW or GST-UvsW-K141R protein. After incubation for 15 min at 37°C, reactions were terminated by the addition of a solution containing 0.2% sodium dodecyl sulfate, 17 mM EDTA, 13% glycerol, and proteinase K (200 µg/ml). Reaction products were separated by electrophoresis through a 0.8% agarose gel with 1× TBE as the running buffer. The agarose gels were dried, and the products were visualized by autoradiography.

	RESULTS

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Characterization of uvsW-K141R phage. An amino acid substitution at position 141 (KR) renders UvsW null for DNA-dependent ATPase and DNA helicase activities (3). Previous in vivo characterization of UvsW-K141R had only been performed using a plasmid that overexpresses the protein. We therefore began by substituting the uvsW-K141R mutation into the correct location in the phage genome by marker rescue from a plasmid, which involves homologous recombination, and then testing the well-characterized uvsW mutant phenotypes.

View larger version (63K): [in this window] [in a new window]   FIG. 1.   DNA arrest phenotype is suppressed in a K10-uvsW-K141R phage infection. Total DNA was isolated from E. coli BL21 cells infected with K10 (lanes 1 and 5), K10-116 uvsW-K141R (lanes 2 and 6), K10-116 uvsW1 (lanes 3 and 7), or K10-116 (uvsY, lanes 4 and 8) for 5 min (lanes 1 through 4) or 45 min (lanes 5 through 8); digested with SspI; and analyzed on a 0.8% agarose gel, which was stained with ethidium bromide. The size markers were generated from XbaI-digested T4dC DNA.

Replication intermediates formed at ori(uvsY) persist during a uvsW-K141R infection. Two-dimensional gel analysis of the T4 ori(uvsY) region previously revealed a novel "comet" on the Y-arc (1). The comet consists of an accumulation of simple Y molecules with branch points in the region of the ori(uvsY) transcript. Formation of the comet was dependent on both the presence of the origin of replication and DNA synthesis. Various results implied that the Y molecules in the comet consist of intermediates in which unidirectional replication has been primed from the RNA of the origin R-loop. The fork thus formed has progressed out of the restriction fragment, but the second fork for bidirectional replication has not yet begun (1).

uvsW1

uvsW1

View larger version (78K): [in this window] [in a new window]   FIG. 2.   The replicative intermediates generated at ori(uvsY) persist for a longer time during a K10-uvsW-K141R infection. Total genomic DNA was isolated from infections with phage K10 (A) and K10-uvsW-K141R (B) at the indicated times (minutes postinfection). All DNA samples were digested with PacI and SwaI prior to electrophoresis. The first dimension of the two-dimension gel is represented horizontally (left to right) and the second dimension vertically (top to bottom). The gel was subjected to Southern blotting and probed with a DNA fragment that lies within ori(uvsY). Arrows indicate the comet on the simple Y-arc.

View larger version (63K): [in this window] [in a new window]   FIG. 3.   uvsW and uvsY transcription are unaffected in the K10-uvsW-K141R infection. RNA was isolated from the same time courses as those for Fig. 2, separated by electrophoresis through a 1% denaturing (formaldehyde) agarose gel, and probed with specific oligonucleotides (see Materials and Methods) to detect the uvsW (A) and ori(uvsY) (B) transcripts. The infecting phage was K10 (left panels) or K10-uvsW-K141R (right panels). In panel A, the arrow represents the full-length uvsW transcript. In panel B, a faint but longer uvsY transcript was also detected (data not shown), consistent with the findings of Gruidl et al. (11). The RNA markers were from Promega, Inc. nt, nucleotides.

Early expression of UvsW represses replication initiation at ori(uvsY). A previous study demonstrated that inappropriate early expression of UvsW blocked replication of an ori(uvsY)-containing plasmid during T4 infection (3). If UvsW represses T4 chromosomal origin function by unwinding the origin R-loop, then the early expression of UvsW should also abolish comet formation.

uvsW2

View larger version (71K): [in this window] [in a new window]   FIG. 4.   ori(uvsY) is repressed by the inappropriate early expression of uvsW. E. coli cells harboring plasmid pKCK41 (uvsW+) (A and B) or pKCD444 (uvsW2) (C) were either induced with IPTG (B and C) or were not induced (A) prior to infection with phage strain K10. DNA samples were prepared at the indicated times postinfection and were analyzed by two-dimensional gel electrophoresis as described for Fig. 2. The arrows indicate the comet region on the simple Y-arc.

View larger version (78K): [in this window] [in a new window]   FIG. 5.   The inappropriate early expression of uvsW mutant protein alters replication from ori(uvsY). E. coli cells harboring plasmid pKCK43 (uvsW-K141R) (A), pKCK42 (uvsW with large inversion) (B), or pKCD444 (uvsW2) (C) were induced with IPTG prior to infection with phage K10. DNA samples were prepared at the indicated time points and analyzed by two-dimensional gel electrophoresis as described for Fig. 2. The arrows indicate the comet region on the simple Y-arc.

Generation of a synthetic R-loop at ori(uvsY). The above in vivo data strongly support our hypothesis that UvsW represses initiation of replication at ori(uvsY). Since UvsW has been shown to be a DNA helicase (3), we speculated that UvsW represses replication by unwinding the origin transcript from ori(uvsY). In order to test this model directly, we created a synthetic R-loop at ori(uvsY). The R-loop was formed by annealing an ori(uvsY)-specific transcript, generated with T7 RNAP, to a supercoiled plasmid pKK405, which harbors the T4 origin (see Materials and Methods). A stable R-loop was formed on supercoiled, but not linear, pKK405 plasmid (Fig. 6A, lanes 1 and 2, respectively), suggesting that negative superhelicity is necessary to stabilize the R-loop. As expected, R-loops were not formed on supercoiled pBR322, arguing that the RNA specifically hybridizes to ori(uvsY) in pKK405 (Fig. 6A, lane 3). Linearization of the R-loop plasmid by restriction enzyme digestion (at sites distal to the RNA) resulted in the loss of the RNA from the input plasmid DNA (data not shown), again consistent with negative superhelicity stabilizing the R-loop by favoring the unwound state of the two DNA strands.

View larger version (20K): [in this window] [in a new window]   FIG. 6.   A synthetic R-loop can be formed at ori(uvsY). (A) The formation of an R-loop was tested on supercoiled (lane 1) or linearized (lane 2) pKK405 [pBR322 harboring ori(uvsY)] or supercoiled pBR322 (lane 3) at a ratio of 100 pmol of 32P-labeled RNA to 10 pmol of DNA. Products were separated by electrophoresis through a 0.8% agarose gel and visualized by autoradiography. The R-loop migrates at the position of free plasmid DNA in the agarose gel (data not shown). The positions of the free RNA (arrow) and R-loop (*) are indicated. (B) Mapping of RNA to ori(uvsY). R-loop was generated with 32P-labeled RNA, purified away from free RNA, and treated with HindIII after fixation of the displaced strand with glyoxal (see Materials and Methods). The untreated R-loop is shown in lane 1, R-loop treated with glyoxal only is shown in lane 2, and R-loop treated with glyoxal and HindIII is shown in lane 3. HindIII cleaves pKK405 into a 4.4-kb vector fragment and a 1.38-kb ori(uvsY) insert, and only the latter contained the RNA label. The size markers were generated from XbaI-digested T4dC DNA. (C) R-loop formation was optimized by varying the ratio of RNA to DNA during the assembly protocol. The samples were treated with BglII, which cleaves free DNA but not R-loops (see Materials and Methods). The linear (l; cleaved) and supercoiled (sc; uncleaved) products were separated by electrophoresis through a 0.8% agarose gel and visualized by Southern blotting. Maximal R-loop formation was obtained at a ratio of 100 pmol of RNA to 10 pmol of DNA.

UvsW can unwind RNA from a synthetic R-loop. The synthetic R-loop provided a substrate to test the potential RNA-DNA helicase activity of UvsW. Purified GST-UvsW fusion protein was previously shown to be active in both ATPase and DNA helicase activities, while the mutant GST-UvsW-K141R was found to be inactive in both assays (3). We first measured R-loop dissociation with increasing concentrations of either GST-UvsW or GST-UvsW-K141R proteins in the presence or absence of ATP. Wild-type GST-UvsW dissociated the RNA from the R-loop in an ATP-dependent reaction (Fig. 7A, compare lanes 1 through 4 to lanes 5 through 8). As with the previous DNA helicase assays, the GST-UvsW-K141R protein was completely inactive (Fig. 7B). Because dissociation of RNA from the R-loop by GST-UvsW was dependent on ATP (Fig. 7A) and MgCl₂ (data not shown), the dissociation is apparently a true helicase activity and not just a helix destabilizing activity.

View larger version (39K): [in this window] [in a new window]   FIG. 7.   UvsW displays an RNA-DNA helicase activity. Unwinding reaction mixtures contained 80 fmol of radioactive R-loop and the following increasing amounts of either GST-UvsW (A) or GST-UvsW-K141R (B): 57 fmol (lanes 1 and 5); 115 fmol (lanes 2 and 6); 217 fmol (lanes 3 and 7); and 576 fmol (lanes 4 and 8). ATP was absent in lanes 1 through 4 and was present in lanes 5 through 8. All products were separated by electrophoresis through a 0.8% agarose gel and visualized by autoradiography. The positions of the free RNA (arrow) and R-loop (*) are indicated.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this study we have analyzed the role of the T4 UvsW protein in the repression of origin-dependent replication at late times of infection. Based on our results, we conclude that UvsW represses T4 origins by removing the RNA from origin R-loops via an RNA-DNA helicase activity. First, the origin replicative intermediates disappear much faster in a wild-type infection (uvsW⁺) than in a uvsW mutant infection. In the wild-type infection, the disappearance of the replicative intermediates correlated closely with the appearance of the uvsW mRNA. Second, the expression of wild-type UvsW protein prior to T4 infection prevented formation of replicative intermediates at the T4 origin. Third, purified GST-UvsW but not GST-UvsW-K141R protein effectively unwound the RNA from a synthetic origin R-loop. These results provide strong evidence to distinguish between two previous models: (i) origins are repressed at the transcriptional level by modification of the host RNAP into its late form (28), and (ii) UvsW actively represses origin function (3, 7).

Our two-dimensional gel analysis of wild-type and uvsW mutant infections, in conjunction with Northern blot analysis, strongly argues that the temporal regulation of gene expression does not play a significant role in repressing origin-dependent replication in the wild-type infection. The temporal appearance of the uvsY (middle) and uvsW (late) transcripts was identical between the uvsW⁺ and uvsW infections, indicating that the regulation of gene expression was the same in both infections. As mentioned above, the comet, consisting of accumulated replicative intermediates, disappeared just as the late uvsW message was increasing in the wild-type infection. Thereafter, the comet was absent for the remainder of the infection, even though origin transcripts were still abundant for some time. If not for the production of UvsW protein, these origin transcripts that are present at late times could be capable of initiating replication, as evidenced by the prolonged appearance of the comet in the uvsW mutant infection. Indeed, the temporal appearance of the comet in the uvsW mutant infection roughly paralleled that of the origin transcript. At present, we do not know if origin transcripts are still being actively synthesized at these late times, indicating a mixture of middle- and late-mode RNAP in the cell, or if middle-mode transcription is shut off but the origin transcripts have a long half-life.

We found a condensed comet that persisted for a longer time when UvsW-K141R was overexpressed prior to T4 infection. The comet in the wild-type infection consists of replicative intermediates in which unidirectional replication has been primed from the RNA of the origin R-loop (1). The fork thus formed has progressed out of the restriction fragment, but the leftward fork for bidirectional replication has not yet begun. The comet was also condensed into a spot in an RNase H-deficient infection, arguing that RNase H often processes the 5' end of the origin transcript in the wild-type infection (1). The generation of the condensed comet with overexpressed UvsW-K141R therefore suggests that binding of UvsW-K141R to the R-loop prevents 5'-end processing by RNase H. A model to explain the persistence of the comet is that the overproduced UvsW-K141R protein also prevents R-loop unwinding by the wild-type protein made from the infecting phage genome.

The two-dimensional gel analysis is limited in defining the precise role of UvsW at the origin, since it does not assay R-loops directly but rather provides a snapshot of origin replication intermediates. The development of the synthetic R-loop at ori(uvsY) allows us to mimic the initial intermediate of replication during in vitro experiments. This R-loop has recently been used as a substrate for in vitro replication assays using purified T4 proteins (31a). The RNA transcript served as a very efficient primer for leading-strand synthesis, strongly validating previous in vivo data. The unwinding experiments described here demonstrate that UvsW can dissociate the RNA from the R-loop substrate via RNA-DNA helicase activity, providing biochemical evidence that UvsW represses the origins by removing the RNA from the origin R-loop.

The E. coli RecG protein is a functional analog of UvsW. Both proteins are involved in recombination and repair and demonstrate helicase activity on branched DNA substrates (3, 27). Like UvsW, RecG has been shown to inhibit replication and unwind R-loops (10, 32, 36). Thus, overexpression of RecG reduces the copy number of plasmids harboring the ColE1 replicon, and the ability of RecG to unwind R-loops is correlated to its ability to reduce copy number. Futhermore, an E. coli recG mutant, like an rnhA mutant, displays constitutive stable DNA replication (cSDR, oriC-independent replication), which is thought to initiate from artificially stabilized R-loops (13, 15). Although E. coli recG or rnhA single mutants survive, the double mutant is nonviable, indicating that accumulation of excess R-loops somehow leads to cell death (13). The strongest evidence that UvsW is a functional analog of E. coli RecG is that the growth of the recG rnhA double mutant can be rescued by the expression of UvsW (3). With the results described here, we conclude that UvsW rescues the double mutant by alleviating the accumulation of R-loops. It is interesting to speculate that replication from R-loop substrates is a primordial replication system which has become repressed in most systems, perhaps because it is difficult to coordinate with cell division and growth.

Another interesting point is that RecG, which is present prior to T4 infection, does not block origin R-loop formation during T4 infection. Synthesis of new RecG protein is presumably shut off upon T4 infection through the arrest of host transcription and translation (for a review, see reference 23). Perhaps pre-existing RecG protein is also inactivated, for example, by covalent modification. Another possibility is that coating of the displaced single strand of the R-loop by the T4-encoded single-strand DNA binding protein, gp32, blocks RecG from binding (but does not interfere with binding and unwinding by UvsW).

One of the most important phenotypes of a uvsW mutant phage is that the arrested DNA synthesis normally caused by mutations that block recombination-dependent replication is restored (5, 40, 41). Previous experiments using plasmid model systems indicated that a uvsW mutation does not restore the arrested DNA synthesis by directly rescuing recombination-dependent replication but rather activates an alternative mode of late replication involving replication origins (7). Based on our analysis of ori(uvsY) here, this alternative mode of replication could simply depend on the persistence of R-loops at the known T4 origins even after middle-mode transcription is blocked (i.e., long-lived origin transcripts). In addition, the UvsW protein may normally remove RNA from R-loops at late promoters, which would thus qualify as cryptic origins that are only active in uvsW mutant infections.

In summary, the conclusion of this study is that UvsW, a protein expressed late during T4 infection, is an RNA-DNA helicase that represses origin-dependent replication by unwinding R-loops. In addition to repressing origin DNA replication, UvsW very likely promotes the process of recombination-dependent replication that becomes dominant at late times of infection (for a review, see reference 20). Thus, UvsW functions as a molecular switch, changing the substrate for T4 replication from R-loops to D-loops. Although the precise role of UvsW in recombination-dependent replication is not known, one reasonable model is that the protein promotes strand invasion to form D-loops.