Proc. Nati. Acad. Sci. USA Vol. 90, pp. 1315-1319, February 1993 Biochemistry RuvA and RuvB proteins of exhibit DNA 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of RuvAB-Holliday length), we show that RuvA and RuvB promote strand dis- junction complexes (C.A. Parsons and S.C.W., unpublished placement with a 5' -b 3' polarity.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 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 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 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. DNA was reaction completion min. visualized by autoradiography on Kodak XAR film. The and cofactor requirements are therefore similar to those percentage offragment displaced from the annealed substrate observed with RuvAB-mediated dissociation of synthetic was quantitated with a laser densitometer (Molecular Dy- Holliday junctions (19). namics model 300). 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 experi- 2 rnM ATP ment in which RuvA was varied from 0 to 100 nM with RuvB 15 mM MgCIl2 at 60 nM (at 1 AM DNA), the percentage of displaced fragment increased sharply between 1 and 20 nM RuvA and m then reached a plateau (Fig. 3A). In related experiments (data :>i 30 riM RLvA '20 nl!M RuvB not shown), we observed that the amount of RuvA required

c to saturate the reaction was independent of the RuvB con- 0 centration (60 and 625 nM). However, more RuvA was _r_.. Cf) + cm M. CL .1 100 V) .r; > > J. _7, C( D =S l CE tD 'D' a_ , L-C -_._ D < < _D CD o¢ C) N 0 Wl 'f) +C+ -1 + 4- 80

66 nt R 60 m ~ qmd-wo-w E 40 -t 10) 20/ 0

FIG. 1. Helicase activity of RuvA and RuvB proteins. Reaction 0 10 20 30 mixtures contained 1 AM DNA substrate (ssDNA with annealed 32P-labeled 66-nt fragment) in helicase buffer (lanes a-g), or in buffer time (min) without ATP (lanes h-m). For reactions of lanes i-m, the buffer contained 2 mM ADP (lane i), 1 mM adenosine 5'-[y-thio]triphos- FIG. 2. Time course of the strand displacement reaction. A phate (ATP[yS]) (lanej), 1 mM ATP (lane k), 1 mM ATP plus 0.5 mM large-scale reaction (180 Al) containing 2 1AM substrate (ssDNA with ATP[yS] (lane 1), or 1 mM ATP plus 5 mM ADP (lane m). For lane annealed 32P-labeled 52-nt fragment) in helicase buffer was incubated n, the reaction mixture contained helicase buffer without MgCl2. for 5 min at 370C, prior to addition ofpre-mixed RuvA and RuvB (0.67 RuvA and/or RuvB were present as indicated and incubation was for ,uM and 1.34 jLM, respectively). At the times indicated after protein 25 min at 37°C. Reactions were stopped and products were analyzed addition, 20-AI aliquots were stopped and the products were analyzed by 1% agarose gel electrophoresis. A control reaction mixture (lane by agarose gel electrophoresis and quantitated. The first time point b) was heat-denatured at 100°C for 3 min prior to loading. represents a sample stopped after 15 sec. Biochemistry: Tsaneva et al. Proc. Natl. Acad. Sci. USA 90 (1993) 1317 100 A !Duplex length lbpfl 152A 1 '

80 RujvA RLJv CC Substrate ._-_ _ _ (0C 60

0 558 Ft a- E 40 w 337 nt-- 20 0- 197 nt-- 0 *-I I I I I 0 20 40 60 80 100 1220 140 nt- RuvA (nM) B 100 80 (U 52 nt- _ Q V) 60 a) a b c d q K 0) E 40 0) FIG. 4. Length dependence. Annealed substrates (2 AiM) con- taining 32P-labeled fragments of length 52, 140, 197, 337, or 558 nt o0-0- 20 were incubated for 25 min at 37TC in helicase buffer without or with RuvA (0.03 ,M) and RuvB (0.06 AM) as indicated. Products were 0 analyzed by 6% PAGE. For each substrate, a control reaction 0 20 40 60 80 100 120 140 160 180 mixture was heat-denatured prior to loading. RuvB (nM) quired for displacement of the longer annealed fragments FIG. 3. Requirement for RuvA and RuvB proteins. Standard (data not shown). reaction mixtures containing 1 ,uM substrate (ssDNA with annealed In view of the inefficient displacement of longer fragments 32P-labeled 52-nt fragment) and 0.06 jLM RuvB (A) or 0.03 uM RuvA by RuvA and RuvB, we tested the effect of SSB on the (B) were supplemented on ice with RuvA (A) or RuvB (B) to the displacement reaction. Using conditions similar to those indicated concentrations. The mixtures were then incubated for 25 described in Fig. 4, we observed that addition of SSB (7 nM min at 37TC. Reactions were stopped and the products were analyzed tetramers) resulted in a 3-fold stimulation ofthe displacement as described in Fig. 2 legend. Background values of 1.2% (A) and of the 337-nt fragment (data not shown). However, addition 0.7% (B) have been subtracted. ofsaturating amounts of SSB (70 nM) was found to inhibit the needed to saturate the reaction at higher DNA concentrations RuvAB helicase reaction. Polarity of the DNA Helicase. The polarity of the helicase (data not shown). in When the concentration ofRuvA was kept constant (27 nM reaction was determined using the DNA substrates shown substrates were con- for 1 ,uM DNA substrate), the helicase reaction was depen- Fig. 5. The first pair of (la and lb) Pst I 4X174 ssDNA a 5'- or dent on the concentration of RuvB. In this case, the percent- structed by cleavage of carrying age of displaced fragment increased as the RuvB concentra- 3'-end-labeled 66-nt fragment. The resulting linear ssDNA tion increased from 20 to 70 nM, and reached a plateau above carried terminal duplex regions which were 40 and 26 nt in 80 nM RuvB (Fig. 3B). Similar curves were obtained at DNA length. Either the 40-mer (substrate la) or the 26-mer (sub- concentrations of 1-10 AtM (data not shown). strate lb) was 32P-labeled. When substrate la was treated Displacement of Annealed Fragments of Different Lengths. with RuvA and RuvB proteins, little of the 5'-labeled 40-mer In the experiments described above, the DNA helicase was released (Fig. 5, lane b). In contrast, treatment of activity of RuvA and RuvB was demonstrated by using DNA substrate lb resulted 'in the efficient unwinding of the 3'- substrates containing short duplex regions of52 and 66 bp. To labeled 26-mer (lane e). As controls, the substrates were determine the effect of duplex length, we produced a series heat-denatured to release the 40- and 26-mers (lanes c and t). ofannealed substrates carrying 32P-labeled fragments 52, 140, Both substrates la and lb contained a small amount of 197, 337, or 558 nt in length. Each substrate was used in circular DNA (which had resisted Pst I digestion), and reactions with RuvA and RuvB, and the products were release of the 66-mer served as an internal control for analyzed by electrophoresis followed by autoradiography unwinding (lanes b, c and e, f). (Fig. 4). We found that the amount offragment displaced was The results obtained with substrates la and lb indicated a inversely related to the length of the duplex DNA, so that bias toward the release of the 26-mer fragment located at the long fragments were displaced poorly. The efficiency of the 3' end of the single strand. To confirm the polarity of the helicase reaction was greater for the 52- and 66-nt fragments unwinding reaction, a second pair of substrates (2a and 2b) (-90% of the fragments were displaced) compared with the were prepared by Fsp I cleavage of 4X174 ssDNA carrying 140-nt (75%), 197-nt (65%), and 337-nt (30%6) fragments (Fig. a 52-nt-long fragment. In this case, the terminal duplex 4). Only about 10% of the 558-nt fragment was displaced by regions were more closely matched in length (24 and 28 nt). RuvA and RuvB. Again, we observed the preferential release of one fragment. Since earlier experiments (Fig. 3B) indicated that helicase The 28-mer located at the 3' end ofthe linear single strand was activity was related to the concentration of RuvB, we tested efficiently released (Fig. 5, lane k), whereas the 24-mer at the the ability of RuvA and RuvB to promote strand displace- 5' end was only poorly displaced (lane h). ment ofthe various annealed substrates over a range ofRuvB Since the experiments with both substrates showed com- concentrations. We observed that short fragments could be plete displacement of the fragments at the 3' end but only displaced at concentrations of RuvB lower than those re- poor displacement ofthose at the 5' end, we conclude that the 1318 Biochemistry: Tsaneva et al. Proc. Natl. Acad. Sci. USA 90 (1993) substrate 1 substrate 2 lyzable ATP analog, ATP[IyS], could not substitute for ATP Pst PstI FspI Fspl and inhibited the reactions supported by ATP. These results --3' (a) 5 3 (a)a 3' 40 * * indicate that strand displacement, like branch migration (18, 2~6 5' 3' 24 28 5' 19), requires ATP hydrolysis. In this study, no special effort was made to define the 2'* 28 53 (b) 5' =36 5b)b) substrate requirements of the RuvAB DNA helicase. Heli- 40 case activity was observed on circular and linear ssDNA molecules that were partially duplex. We have not observed : la 1b 2a 2b ._. the unwinding offully duplex DNA, even those as short as 50 u -, RuvA V o o bp (ref. 19 and unpublished data). The need for ssDNA most -- - _ 4_ IbRauvB -i _ _R probably reflects the binding affinity of the enzymes, partic- Substrate d .4 dm __w __0 ..ft -- Substrate ularly that of RuvA. In support of this, the amount of RuvA required to saturate the reaction was dependent upon the DNA concentration, consistent with observations which indicate nonspecific binding of RuvA to ssDNA (ref. 20, and unpublished data). Since RuvA shows a high binding affinity for model Holliday junctions (19), it is possible that binding 66 nt uncut to the annealed substrate may be facilitated by secondary 52 nt structures within the ssDNA. uncut Shiba et al. (20) have shown that the ATPase activity of 40 nt gm RuvB is stimulated by the presence of RuvA and DNA, _-- 28 nt suggestive of a direct interaction between the RuvA and 26 nt tat RuvB proteins and ofRuvAB with DNA. Recently, the direct interaction ofRuvA with RuvB was demonstrated by glycerol q. - 24 nt gradient centrifugation (22), and RuvAB-Holliday junction complexes formed in the presence ofATP[yS] were detected by band-shift assays (C.A. Parsons, and S.C.W., unpub- a b c d e f g h j k lished data). Sequence comparisons indicate that RuvB con- tains GxGKT (26) and DExH motifs (Fig. 6), which are FIG. 5. Polarity of strand displacement promoted by RuvA and RuvB. The two helicase substrates were derived from circular 4X174 common features shared by a number of ATP-dependent ssDNA carrying a 66- or 52-nt fragment and were produced by DNA helicases from E. coli (27-30). It is therefore likely that digestion with Pst I (substrate 1) or Fsp I (substrate 2). Each RuvB provides the motive force for strand displacement, substrate was 32P-labeled at the 5' (substrates la and 2a) or 3' whereas RuvA functions to bind the DNA substrate and/or (substrates lb and 2b) terminus of the annealed fragment, as indi- to form a functional complex. cated by stars. Reaction mixtures containing 2 ILM DNA substrate Although short annealed fragments were efficiently dis- were incubated in the absence or presence of RuvA (0.11 AM) and placed by RuvA and RuvB, long fragments were displaced RuvB (0.24 .uM) for 30 min at 370C. For each substrate, a control poorly. However, the helicase assay used here detects only reaction mixture was heat-denatured prior to loading. Products were complete displacement, and partial unwinding events fol- analyzed by 12% PAGE. lowed by reannealing would remain undetected. The in- creased efficiency of displacement of a 337-nt fragment by RuvAB-mediated strand displacement reaction has a 5'-+ 3' low concentrations of SSB is suggestive of partial unwinding polarity (defined relative to the ssDNA). and reannealing events. Interestingly, we found that the displacement of longer fragments required higher RuvB con- DISCUSSION centrations than that required for short annealed fragments. We have shown that the RuvA and RuvB proteins, which These results indicate that the poor displacement of the promote the branch migration of Holliday junctions in vitro, longer fragments may be due to low helicase processivity. exhibit DNA helicase activity on partially duplex DNA Alternatively, the proteins may be engaged in a process substrates. This helicase activity requires the action of both involving a short patch of duplex unwinding and reannealing proteins and an energy cofactor such as ATP. A nonhydro- ("bubbling"). This could be highly processive, but in the case

tif I (ATP-binding) Ililtit 1I

RuvB HL L I FGP P L GKTTLAN IV V L F I D ELI 11 R L S P L.vrIB HQT L L GV T S GKT FT I AN V D G L 'VVVDFE:SH1IV TI P '4 UvrD) NLLV L AGA S GK T RV L VH R '14 F T N I 1,V D F I)D T NN RecB ER L I EA S A T GKT F T 1 AA L F P V A M I D E F Q I) T D) P ReeD I S VI S GG P T GKT TT VAK L V L V V D F A S M I I) L RecQ I) C L V V M P T G GK S IC YQ I P P V l L AV D ElA 11 C I S () I lcD MS LLV L A GTA S GK T S V L VA R -'11 W K II 11 V D EF Q L) I S E) Rep P C LV L AG A S GKTRV I TNK NlpI R Y LLV D E!Y()I) TNN Tral FFTVVQGY A v G K T TQ F R A V ""sN T 1 FLLL) F. S S MI V CN

G x G K 1- 1) [-. s I.. s

FIG. 6. Comparison of the RuvB sequence with those of other E. coli helicases. Two consensus domains characteristic of DNA helicases are boxed (27, 28). The numbers on the left indicate the positions where the alignments are initiated. Identical amino acids and conservative changes are indicated by the dark and light shadings, respectively. Sequence data were taken from the Swiss-Prot data base (Release 22). Biochemistry: Tsaneva et al. Proc. Natl. Acad. Sci. USA 90 (1993) 1319

of the longer duplex substrates would lead to a lower prob- 3. Oh, E. K. & Grossman, L. (1987) Proc. Natl. Acad. Sci. USA ability of full displacement events. It is not known whether 84, 3638-3642. RuvB, or the RuvAB complex, is able to translocate along 4. Orren, D. K., Selby, C. P., Hearst, J. E. & Sancar, A. (1992) DNA, as has been observed with the UvrAB complex (31). J. Biol. Chem. 267, 780-788. Unidirectional translocation along DNA in a 5' 3' direction 5. Taylor, A. F. & Smith, G. R. (1980) Cell 22, 447-457. 6. Dixon, D. A. & Kowalczykowski, S. C. (1991) Cell 66, 361- could be one possible mechanism to explain the polarity of 371. the RuvAB helicase. 7. Umezu, K., Nakayama, K. & Nakayama, H. (1990) Proc. Natl. Previously, it was shown that the RuvAB proteins cata- Acad. Sci. USA 87, 5363-5367. lyzed branch migration in vitro (18-20), a role consistent with 8. West, S. C., Cassuto, E. & Howard-Flanders, P. (1981) Nature the genetic phenotype of ruvA or ruvB mutants (15, 17, 32). (London) 290, 29-33. The observations presented here which show that the ruvA 9. Howard-Flanders, P., West, S. C. & Stasiak, A. J. (1984) and ruvB gene products together exhibit DNA helicase ac- Nature (London) 309, 215-220. tivity may provide insight into the mechanics of the branch 10. Hsieh, P., Camerini-Otero, C. S. & Camerini-Otero, R. D. migration reaction promoted by RuvA and RuvB. The pos- (1990) Genes Dev. 4, 1951-1963. sibility that DNA helicases could play a role in branch 11. Rao, B. J., Jwang, B. & Radding, C. M. (1990) J. Mol. Biol. 213, 789-809. migration was first suggested by studies with the Dda helicase 12. Rao, B. J., Dutreix, M. & Radding, C. M. (1991) Proc. Natl. of bacteriophage T4 (33). However, in this case, Dda protein Acad. Sci. USA 88, 2984-2988. stimulated branch migration catalyzed by the UvsX protein 13. West, S. C. & Howard-Flanders, P. (1984) Cell 37, 683-691. but was unable to promote branch migration alone. Indeed, 14. Muller, B., Burdett, I. & West, S. C. (1992) EMBO J. 11, stimulation ofbranch migration was indirect and occurred as 2685-2693. Dda facilitated the directional assembly of the UvsX protein 15. Lloyd, R. G., Benson, F. E. & Shurvinton, C. E. (1984) Mol. filament (33). The situation with RuvA and RuvB is quite Gen. Genet. 194, 303-309. different, since RuvA and RuvB promote efficient branch 16. Sharples, G. J., Benson, F. E., Illing, G. T. & Lloyd, R. G. migration in the absence of RecA (18, 19). The affinity of (1990) Mol. Gen. Genet. 221, 219-226. 17. Benson, F., Collier, S. & Lloyd, R. G. (1991) Mol. Gen. Genet. RuvA for model Holliday structures indicates that RuvAB 225, 266-272. interacts directly with thejunction and that branch migration 18. Tsaneva, I. R., Miller, B. & West, S. C. (1992) Cell 69, may occur via a coupled reaction in which two duplexes 1171-1180. become unwound at the junction point, thus allowing the 19. Parsons, C. A., Tsaneva, I., Lloyd, R. G. & West, S. C. (1992) exchange of single strands by reannealing. The nearly stoi- Proc. Natl. Acad. Sci. USA 89, 5452-5456. chiometric requirement for RuvB and need for ATPase 20. Shiba, T., Iwasaki, H., Nakata, A. & Shinagawa, H. (1991) activity indicate that RuvAB exploits the hydrolysis of ATP Proc. Natl. Acad. Sci. USA 88, 8445-8449. for separating DNA strands rather than for recycling the 21. Iwasaki, H., Shiba, T., Makino, K., Nakata, A. & Shinagawa, enzyme. Other DNA helicases, such as Dda protein and H. (1989) J. Bacteriol. 171, 5276-5280. 22. Shiba, T., Iwasaki, H., Nakata, A. & Shinagawa, H. (1993) DNA helicase II, are also required in stoichiometric amounts Mol. Gen. Genet., in press. and contrast with DNA helicase I or Rep protein, which act 23. Lloyd, R. G. (1991) J. Bacteriol. 173, 5414-5418. catalytically (1). 24. Tsaneva, I. R., Illing, G. T., Lloyd, R. G. & West, S. C. (1992) Recent work from the Lloyd laboratory has shown that the Mol. Gen. Genet. 235, 1-10. E. coli recG gene product, RecG, can also promote branch 25. Lloyd, R. G. & Buckman, C. (1991) J. Bacteriol. 173, 1004- migration in vitro (34), consistent with genetic data indicating 1011. that RuvAB and RecG are functionally analogous (23). Ex- 26. Benson, F. E., Illing, G. T., Sharples, G. J. & Lloyd, R. G. amination of the RecG sequence has also revealed structural (1988) Nucleic Acids Res. 16, 1541-1550. motifs indicative of a DNA helicase (25). Taken together, 27. Hodgman, T. C. (1988) Nature (London) 333, 22-23. 28. Gorbalenya, A. E., Koonin, E. V., Donchenko, A. P. & Bli- these results may indicate a subset of DNA helicases that nov, V. M. (1989) Nucleic Acids Res. 17, 4713-4730. have evolved to promote the branch migration of Holliday 29. Walker, J. E., Saraste, M., Runswick, M. J. & Gay, N. J. junctions. (1982) EMBO J. 1, 945-951. 30. Gorbalenya, A. E., Koonin, E. V., Donchenko, A. P. & Bli- We thank our colleagues for suggestions and Dr. R. G. Lloyd for nov, V. M. (1988) Nature (London) 333, 22. his advice and communication ofdata prior to publication. B.M. was 31. Koo, H.-S., Claassen, L., Grossman, L. & Liu, L. F. (1991) supported in part by the Swiss National Science Foundation. Proc. Natl. Acad. Sci. USA 88, 1212-1216. 32. Otsuji, N., Iyehara, H. & Hideshima, Y. (1974) J. Bacteriol. 1. Matson, S. W. & Kaiser-Rogers, K. A. (1990) Annu. Rev. 117, 337-344. Biochem. 59, 289-330. 33. Kodadek, T. & Alberts, B. M. (1987) Nature (London) 326, 2. Kornberg, A. & Baker, T. (1992) in DNA Replication (Free- 312-314. man, New York). 34. Lloyd, R. G. & Sharples, G. J. (1993) EMBO J. 12, 17-22.