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Activity in Vitro (Recombination/DNA Repair/Holfiday Junctions/Branch Migration/Strand Exchange) IRINA R

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Activity in Vitro (Recombination/DNA Repair/Holfiday Junctions/Branch Migration/Strand Exchange) IRINA R 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 (receivedfor review September 18, 1992) ABSTRACT The SOS-inducible ruvA and ruvB gene prod- intermediates (15-17). Biochemical studies provided support ucts ofEscherichia coli are required for normal levels ofgenetic for this notion by demonstrating that RuvA and RuvB to- recombination and DNA repair. In vitro, RuvA protein inter- gether promote the branch migration ofHollidayjunctions in acts specifically with Holliday junctions and, together with vitro, leading to the formation of heteroduplex DNA (14, RuvB (an ATPase), promotes their movement along DNA. This 18-20). The way in which RuvA binds specifically to syn- process, known as branch migration, is important for the thetic Hollidayjunctions (19) led us to propose that it targets formation of heteroduplex DNA. In this paper, we show that the RuvB ATPase (21) to the junction where it provides the the RuvA and RuvB proteins promote the unwinding of motor for branch migration (18, 19). Recently, the direct partially duplex DNA. Using single-stranded circular DNA interaction of RuvA and RuvB has been demonstrated both substrates with annealed fragments (52-558 nucleotides in in solution (22) and by the formation of RuvAB-Holliday length), we show that RuvA and RuvB promote strand dis- junction complexes (C.A. Parsons and S.C.W., unpublished placement with a 5' -b 3' polarity. The reaction is ATP- data). dependent and its efficiency is inversely related to the length of In the present work, we show that the RuvA and RuvB the duplex DNA. These results show that the ruvA and ruvB proteins possess DNA helicase activity. We suggest that genes encode a DNA helicase that specifically recognizes Hol- RuvAB-mediated branch migration ofa Hollidayjunction may liday junctions and promotes branch migration. occur by the localized denaturation and reannealing of DNA. DNA helicases play essential roles in DNA replication, MATERIALS AND METHODS repair, and recombination (for review see ref. 1). In bacteria, Proteins. RuvA and RuvB proteins were purified from helicases such as Rep, DnaB, and PriA (n') act at the overexpression vectors as described elsewhere (24). Protein replication fork, where they unwind DNA during replication concentrations were determined by the Lowry (Sigma pro- (2). Unwinding occurs with a defined polarity and is driven at tein assay kit) and Bradford (Bio-Rad protein assay kit) the expense of nucleoside triphosphate hydrolysis. In DNA methods using bovine serum albumin as standard and were repair, the UvrA and UvrB proteins, part of the UvrABC confirmed by spectrophotometry. Amounts of protein are excision nuclease complex, exhibit helicase activity during expressed in moles of monomer. In previous studies (14, 18, the recognition of DNA lesions (3), while UvrD (DNA 19, 24), we used the Bradford assay with ovalbumin as helicase II) is involved in the disassembly of post-incision standard, but this standard leads to an overestimate of complexes (4). protein concentration. E. coli single-stranded-DNA-binding During genetic recombination in Escherichia coli, the for- protein (SSB) was purchased from Pharmacia. mation of recombinant DNA molecules occurs via a series of Oligonucleotides. The 52- and 66-mers were synthesized by well-defined, yet overlapping steps, several of which involve phosphoramidite chemistry on an Applied Biosystems 380B the action of DNA helicases. For example, RecBCD enzyme DNA synthesizer and purified by reverse-phase HPLC. unwinds duplex DNA leading to the initiation of recombina- When necessary, they were further purified by PAGE. tion by RecA protein (5, 6). A similar role is likely to be played Helicase Substrates. DNA substrates were prepared in two by the RecQ helicase (7). In early studies with RecA protein, ways. (i) Oligonucleotides (52 or 66 nt long) were annealed it was thought that the mechanisms ofhomologous pairing and with 4X174 virion DNA. The 52-mer was complementary to strand exchange might involve strand separation. However, 4X174 DNA at nt 130-181 and the 66-mer was complemen- this was not the case (8) and current work indicates the tary to nt 5357-36. (ii) Duplex restriction fragments (140-bp formation of multistranded DNA helices within the RecA Ava II-Dra III, 197-bp Dra III-Pst I, 337-bp Ava II-Pst I, or filament (9-14). Nevertheless, the concept that subsequent 558-bp Stu I-Ava II) were produced by restriction digestion branch migration of a Holliday junction and the formation of of4X174 replicative form I (RFI) DNA and were purified by extensive lengths ofheteroduplex DNA might be catalyzed by sucrose gradient centrifugation and/or by nondenaturing 6% a helicase-like activity remains attractive. PAGE followed by electroelution. They were then denatured In recent studies we focused our attention on the proteins and annealed with 4X174 single-stranded DNA (ssDNA). encoded by the ruv locus of the E. coli chromosome. The Unless stated otherwise, oligonucleotides or restriction RuvA and RuvB proteins interact with each other and fragments were mixed with 20 gg of 46X174 ssDNA at a 1:1 catalyze reactions that are important for genetic recombina- ratio (molecule per molecule) in 50-100 ,ul of10 mM Tris HC1, tion and the recombinational repair of DNA damage. Early pH 7.5/10 mM MgCl2/50 mM NaCl, heated for 3 min at genetic studies showed that ruvA and ruvB mutants had 100'C, incubated for 30 min at 680C, and slowly cooled to similar phenotypes characteristic of a defect in a late step of room temperature. When subsequent restriction digestion recombination, such as the processing of recombination was required, reannealing was carried out in restriction enzyme buffer. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: ssDNA, single-stranded DNA; SSB, ssDNA-binding in accordance with 18 U.S.C. §1734 solely to indicate this fact. protein; ATP[yS], adenosine 5'-[y-thio]triphosphate. 1315 1316 Biochemistry: Tsaneva et al. Proc. Natl. Acad. Sci. USA 90 (1993) For some experiments, oligonucleotides were 5'- or 3'- RESULTS end-labeled prior to reannealing by using [y-32P]ATP and Unwinding of DNA by the RuvA and RuvB Proteins. To test polynucleotide kinase or [a-32P]ddATP and terminal deoxy- the RuvA and RuvB proteins for DNA helicase activity, we nucleotidyltransferase. Alternatively, unlabeled oligonucle- used a simple gel electrophoretic assay which measures the otides or fragments were used and annealed substrates were displacement of a short 32P-labeled oligonucleotide (66 nt labeled in reannealing buffer by addition of 1 mM dithiothrei- long) from single-stranded circular 4X174 DNA. Purified tol, 50 OCi (1 OCi = 37 kBq) of the appropriate [a-32P]dNTP, RuvA and RuvB catalyzed the unwinding of the oligonucle- and 8 units of the Klenow fragment of DNA polymerase I, otide from the single-stranded circle (Fig. 1, lane e). Neither followed by incubation for 20 min at 20TC. RuvA (lanes c and d) nor RuvB (lanes f and g) alone were Annealed substrates were purified by centrifugation capable of unwinding, even at much higher protein concen- through 5-20% sucrose gradients at 42,000 rpm in an SW 50.1 trations. Assays of activity during purification of RuvA and Beckman rotor for 3 hr at 40C. DNA was dialyzed against 10 RuvB indicated that helicase activity peaked with the RuvA mM Tris-HCl, pH 7.5/0.1 mM EDTA/0.1 M NaCl and the and RuvB elution profiles (data not shown). These results concentration was determined from the absorbance at 260 indicate that DNA helicase activity is an intrinsic property of nm. Amounts ofDNA are expressed in moles of nucleotides. the combined action of RuvA and RuvB. Helicase Assay. Unless stated otherwise, reaction mixtures Reaction Requirements and ATP Dependence. The strand (20 Al) contained 0.4-2 ALM annealed substrate DNA in displacement reaction required ATP and Mg2+ (Fig. 1, lanes h helicase buffer (20 mM Tris1HCl, pH 7.5/15 mM MgCl2/2 and n) and was not detected when ATP was replaced by ADP mM ATP/2 mM dithiothreitol with 100 ,g ofBSA per ml, and (lane i) or the nonhydrolyzable ATP analog ATP[(yS] (lane j). 10-50 mM NaCl). RuvA and RuvB (in a volume of 2 ,l) were Concentrations of ATP - 0.5 mM and MgCl2 at 10-20 mM added as indicated. Reactions were stopped and protein was were optimal for activity (data not shown). In the presence of removed by addition of 5 ,l of 5 x stop buffer (0.5% protein- 1 mM ATP, the reaction was partially inhibited by addition of ase K/100 mM Tris HCI, pH 7.5/200 mM EDTA/2.5% SDS), 0.5 mM ATP[-yS] (lane 1) or 5 mM ADP (lane m). followed by incubation at 37°C for 10 min. Products were A time course ofthe RuvAB-mediated strand displacement analyzed by electrophoresis in 1% agarose gels with 40 mM reaction is shown in Fig. 2. In this and following experiments, Tris acetate, pH 8.0/1 mM EDTA as the buffer system. Gels the percent of fragment unwound was determined by laser were dried on Whatman DE81 paper. In some experiments, densitometry following autoradiography. We found that 60%6 products were analyzed by electrophoresis in polyacrylamide of the labeled fragments were displaced after 5 min and the gels run in 89 mM Tris borate, pH 8.3/2 mM EDTA and the went to within 10 The time course gels were dried onto Whatman 3MM paper.
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