IRINA R. TSANEVA, BERNDT MULLER, AND STEPHEN C. WEST. Imperial ..... Tsaneva, I. R., Miller, B. & West, S. C. (1992) Cell 69,. 1171-1180. 19. Parsons ...
Proc. Nati. Acad. Sci. USA Vol. 90, pp. 1315-1319, February 1993 Biochemistry
RuvA and RuvB proteins of Escherichia coli exhibit DNA helicase activity in vitro (recombination/DNA repair/Holfiday junctions/branch migration/strand exchange)
IRINA R. TSANEVA, BERNDT MULLER,
AND
STEPHEN C. WEST
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire, EN6 3LD, United Kingdom
Communicated by Howard A. Nash, November 5, 1992 (received for review September 18, 1992)
ABSTRACT The SOS-inducible ruvA and ruvB gene products ofEscherichia coli are required for normal levels ofgenetic recombination and DNA repair. In vitro, RuvA protein interacts specifically with Holliday junctions and, together with RuvB (an ATPase), promotes their movement along DNA. This process, known as branch migration, is important for the formation of heteroduplex DNA. In this paper, we show that the RuvA and RuvB proteins promote the unwinding of partially duplex DNA. Using single-stranded circular DNA substrates with annealed fragments (52-558 nucleotides in length), we show that RuvA and RuvB promote strand displacement with a 5' -b 3' polarity. The reaction is ATPdependent and its efficiency is inversely related to the length of the duplex DNA. These results show that the ruvA and ruvB genes encode a DNA helicase that specifically recognizes Holliday junctions and promotes branch migration.
DNA helicases play essential roles in DNA replication, repair, and recombination (for review see ref. 1). In bacteria, helicases such as Rep, DnaB, and PriA (n') act at the replication fork, where they unwind DNA during replication (2). Unwinding occurs with a defined polarity and is driven at the expense of nucleoside triphosphate hydrolysis. In DNA repair, the UvrA and UvrB proteins, part of the UvrABC excision nuclease complex, exhibit helicase activity during the recognition of DNA lesions (3), while UvrD (DNA helicase II) is involved in the disassembly of post-incision complexes (4). During genetic recombination in Escherichia coli, the formation of recombinant DNA molecules occurs via a series of well-defined, yet overlapping steps, several of which involve the action of DNA helicases. For example, RecBCD enzyme unwinds duplex DNA leading to the initiation of recombination by RecA protein (5, 6). A similar role is likely to be played by the RecQ helicase (7). In early studies with RecA protein, it was thought that the mechanisms of homologous pairing and strand exchange might involve strand separation. However, this was not the case (8) and current work indicates the formation of multistranded DNA helices within the RecA filament (9-14). Nevertheless, the concept that subsequent branch migration of a Holliday junction and the formation of extensive lengths of heteroduplex DNA might be catalyzed by a helicase-like activity remains attractive. In recent studies we focused our attention on the proteins encoded by the ruv locus of the E. coli chromosome. The RuvA and RuvB proteins interact with each other and catalyze reactions that are important for genetic recombination and the recombinational repair of DNA damage. Early genetic studies showed that ruvA and ruvB mutants had similar phenotypes characteristic of a defect in a late step of recombination, such as the processing of recombination The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
intermediates (15-17). Biochemical studies provided support for this notion by demonstrating that RuvA and RuvB together promote the branch migration of Holliday junctions in vitro, leading to the formation of heteroduplex DNA (14, 18-20). The way in which RuvA binds specifically to synthetic Holliday junctions (19) led us to propose that it targets the RuvB ATPase (21) to the junction where it provides the motor for branch migration (18, 19). Recently, the direct interaction of RuvA and RuvB has been demonstrated both in solution (22) and by the formation of RuvAB-Holliday junction complexes (C.A. Parsons and S.C.W., unpublished data). In the present work, we show that the RuvA and RuvB proteins possess DNA helicase activity. We suggest that RuvAB-mediated branch migration of a Hollidayjunction may occur by the localized denaturation and reannealing of DNA.
MATERIALS AND METHODS Proteins. RuvA and RuvB proteins were purified from overexpression vectors as described elsewhere (24). Protein concentrations were determined by the Lowry (Sigma protein assay kit) and Bradford (Bio-Rad protein assay kit) methods using bovine serum albumin as standard and were confirmed by spectrophotometry. Amounts of protein are expressed in moles of monomer. In previous studies (14, 18, 19, 24), we used the Bradford assay with ovalbumin as standard, but this standard leads to an overestimate of protein concentration. E. coli single-stranded-DNA-binding protein (SSB) was purchased from Pharmacia. Oligonucleotides. The 52- and 66-mers were synthesized by phosphoramidite chemistry on an Applied Biosystems 380B DNA synthesizer and purified by reverse-phase HPLC. When necessary, they were further purified by PAGE. Helicase Substrates. DNA substrates were prepared in two ways. (i) Oligonucleotides (52 or 66 nt long) were annealed with 4X174 virion DNA. The 52-mer was complementary to 4X174 DNA at nt 130-181 and the 66-mer was complementary to nt 5357-36. (ii) Duplex restriction fragments (140-bp Ava II-Dra III, 197-bp Dra III-Pst I, 337-bp Ava II-Pst I, or 558-bp Stu I-Ava II) were produced by restriction digestion of 4X174 replicative form I (RFI) DNA and were purified by sucrose gradient centrifugation and/or by nondenaturing 6% PAGE followed by electroelution. They were then denatured and annealed with 4X174 single-stranded DNA (ssDNA). Unless stated otherwise, oligonucleotides or restriction fragments were mixed with 20 gg of 46X174 ssDNA at a 1:1 ratio (molecule per molecule) in 50-100 ,ul of 10 mM Tris HC1, pH 7.5/10 mM MgCl2/50 mM NaCl, heated for 3 min at 100'C, incubated for 30 min at 680C, and slowly cooled to room temperature. When subsequent restriction digestion was required, reannealing was carried out in restriction enzyme buffer. Abbreviations: ssDNA, single-stranded DNA; SSB, ssDNA-binding protein; ATP[yS], adenosine 5'-[y-thio]triphosphate.
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Proc. Natl. Acad. Sci. USA 90 (1993)
For some experiments, oligonucleotides were 5'- or 3'end-labeled prior to reannealing by using [y-32P]ATP and polynucleotide kinase or [a-32P]ddATP and terminal deoxynucleotidyltransferase. Alternatively, unlabeled oligonucleotides or fragments were used and annealed substrates were labeled in reannealing buffer by addition of 1 mM dithiothreitol, 50 OCi (1 OCi = 37 kBq) of the appropriate [a-32P]dNTP, and 8 units of the Klenow fragment of DNA polymerase I, followed by incubation for 20 min at 20TC. Annealed substrates were purified by centrifugation through 5-20% sucrose gradients at 42,000 rpm in an SW 50.1 Beckman rotor for 3 hr at 40C. DNA was dialyzed against 10 mM Tris-HCl, pH 7.5/0.1 mM EDTA/0.1 M NaCl and the concentration was determined from the absorbance at 260 nm. Amounts of DNA are expressed in moles of nucleotides. Helicase Assay. Unless stated otherwise, reaction mixtures (20 Al) contained 0.4-2 ALM annealed substrate DNA in helicase buffer (20 mM Tris1HCl, pH 7.5/15 mM MgCl2/2 mM ATP/2 mM dithiothreitol with 100 ,g of BSA per ml, and 10-50 mM NaCl). RuvA and RuvB (in a volume of 2 ,l) were added as indicated. Reactions were stopped and protein was removed by addition of 5 ,l of 5 x stop buffer (0.5% proteinase K/100 mM Tris HCI, pH 7.5/200 mM EDTA/2.5% SDS), followed by incubation at 37°C for 10 min. Products were analyzed by electrophoresis in 1% agarose gels with 40 mM Tris acetate, pH 8.0/1 mM EDTA as the buffer system. Gels were dried on Whatman DE81 paper. In some experiments, products were analyzed by electrophoresis in polyacrylamide gels run in 89 mM Tris borate, pH 8.3/2 mM EDTA and the gels were dried onto Whatman 3MM paper. DNA was visualized by autoradiography on Kodak XAR film. The percentage of fragment displaced from the annealed substrate was quantitated with a laser densitometer (Molecular Dynamics model 300). 2 rnM ATP 15 mM MgCIl2
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RESULTS Unwinding of DNA by the RuvA and RuvB Proteins. To test the RuvA and RuvB proteins for DNA helicase activity, we used a simple gel electrophoretic assay which measures the displacement of a short 32P-labeled oligonucleotide (66 nt long) from single-stranded circular 4X174 DNA. Purified RuvA and RuvB catalyzed the unwinding of the oligonucleotide from the single-stranded circle (Fig. 1, lane e). Neither RuvA (lanes c and d) nor RuvB (lanes f and g) alone were capable of unwinding, even at much higher protein concentrations. Assays of activity during purification of RuvA and RuvB indicated that helicase activity peaked with the RuvA and RuvB elution profiles (data not shown). These results indicate that DNA helicase activity is an intrinsic property of the combined action of RuvA and RuvB. Reaction Requirements and ATP Dependence. The strand displacement reaction required ATP and Mg2+ (Fig. 1, lanes h and n) and was not detected when ATP was replaced by ADP (lane i) or the nonhydrolyzable ATP analog ATP[(yS] (lane j). Concentrations of ATP - 0.5 mM and MgCl2 at 10-20 mM were optimal for activity (data not shown). In the presence of 1 mM ATP, the reaction was partially inhibited by addition of 0.5 mM ATP[-yS] (lane 1) or 5 mM ADP (lane m). A time course of the RuvAB-mediated strand displacement reaction is shown in Fig. 2. In this and following experiments, the percent of fragment unwound was determined by laser densitometry following autoradiography. We found that 60%6 of the labeled fragments were displaced after 5 min and the reaction went to completion within 10 min. The time course and cofactor requirements are therefore similar to those observed with RuvAB-mediated dissociation of synthetic Holliday junctions (19). Requirement for RuvA and RuvB. To determine the specific requirement for RuvA and RuvB, the concentration of one was varied while the other was held constant. In an experiment in which RuvA was varied from 0 to 100 nM with RuvB at 60 nM (at 1 AM DNA), the percentage of displaced fragment increased sharply between 1 and 20 nM RuvA and then reached a plateau (Fig. 3A). In related experiments (data not shown), we observed that the amount of RuvA required to saturate the reaction was independent of the RuvB concentration (60 and 625 nM). However, more RuvA was
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