J. Mol. Biol. (1991) 221,

131-145

RecA-mediated Annealing of Single-stranded DNA and Its Relation to the Mechanism of Homologous Recombination Berndt Miiller”? and Andrzej Stasiak1y2$ ‘Institut

Zellbiologie, ETH-Ziirich, CH-8093 Ziirich-HGnggerberg, Switzerland ‘Laboratoire d’dnalyse Ultrastructurale, Bcitiment de Biologie Universite’ de Lausanne, CH-1015 Lausanne-Dorigny, Switzerland

fiir

(Received

27 August

and

1990; accepted 13 May 1991)

We demonstrate that RecA protein can mediate annealing of complementary DNA strands in vitro by at least two different mechanisms. The first annealing mechanism predominates under conditions where RecA protein causes coaggregation of single-stranded DNA (ssDNA) molecules and where RecA-free ssDNA stretches are present on both reaction partners. Under these conditions annealing can take place between locally concentrated protein-free complementary sequences. Other DNA aggregating agents like histone Hl or ethanol stimulate annealing by the same mechanism. The second mechanism of RecA-mediated annealing of complementary DNA strands is best manifested when preformed saturated RecA-ssDNA complexes interact with proteinfree ssDNA. In this case, annealing can occur between the ssDNA strand resident in the complex and the ssDNA strand that interacts with the preformed RecA-ssDNA complex. Here, the action of RecA protein reflects its specific recombination promoting mechanism. This mechanism enables DNA molecules resident in the presynaptic RecA-DNA complexes to be exposed for hydrogen bond formation with DNA molecules contacting the presynaptic RecA-DNA filament.

Keywords: homologous protein-DNA

recombination; RecA protein; DNA annealing; interactions; RecA-DNA complexes

& Lehman, 1987; Griffith & Harris, 1988; Radding, 1988; Stasiak & Egelman, 1988; Rota & Cox, 1990; Kowalczykowski, 1991). The search for homology consists of two distinct steps. The first step is the homology-independent extensive coaggregation of RecA-complexed DNA molecules together with protein-free DNA molecules. In the second step, the enhanced DNA concentration within the coaggregates leads to frequent intermolecular contacts between protein-free DNA molecules and RecA-complexed DNA molecules. In the complexes, the DNA is unwound and stretched, which might facilitate pairing (Stasiak & DiCapua, 1982). After the recognition of homology between some regions of interacting DNA molecules, the protein-free DNA partner becomes enveloped into the RecA-DNA complex and aligned with the DNA molecule residing in the complex. The actual molecular mechanism by which RecA mediates the homologous recognition between interacting DNA molecules is not yet established. Two models for the mechanism are frequently considered (Stasiak & Egelman, 1988). One model called “strand separation before pairing” proposes that

1. Introduction RecA§ is one of the crucial recombination promoting proteins in Escherichia coli. This protein alone can mediate the following three stages of the in vitro recombination: (1) the formation of presynaptic complexes between RecA and partially or completely single-stranded DNA molecules; (2) the search for homologous sequences between the DNA within the presynaptic complexes and protein-free duplex DNA, leading to homologous alignment; and (3) strand exchange and release of DNA products (for a review, see Howard-Flanders et al., 1984; Cox t Present address: Imperial Cancer Research Fund, Glare Hall Laboratories, South Mimms, Herts, EN6 3LD, U.K. $ Author to whom correspondence should be addressed at : Laboratoire d’Analyse Ultrastructurale, BBtiment de Biologie, Universite de Lausanne, CH-1015 Lausanne-Dorigny, Switzerland. $ Abbreviations used: RecA, RecA protein of Escher&&z co&; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; bp, base-pair(s); kb, lo3 bases or base-pairs. 131 0022-2836/S1/170131~15

$03.00/O

0

1991 Academic Press Limited

132

B. Miiller and A. Stasiak

RecA has a strand separating activity that would permit intermolecular subsequent annealing between strands of two interacting DNA molecules. This can lead to homologous recognition by the formation of regular Watson-Crick base-pairs. The second model called “pairing before strand separation” proposes that specific additional hydrogen bonds can lead to recognition of homology without the need to separate the duplex DNA molecules into two ssDNA strands (McGavin, 1977). RecA stimulates annealing of complementary DNA strands in vitro (Weinstock et al., 1979). For RecA-mediated in vitro recombination, the presence of ssDNA in at least one of the reaction partners is required (West et al., 1982). These two observations supported the proposal that the actual mechanism of homologous recognition in RecA-mediated recombination involves annealing between singlestranded regions of two interacting DNA molecules et al.: 1979; Cunningham et al., 1979; (McEntee Radding, 1981). However, there are clear differences between the requirements for efficient in vitro recombination and efficient’ annealing, for suggesting that the two reactions might occur by different mechanisms. In vitro recombination reactions depend on the saturation of one partner DNA molecule with RecA protein (Shibata et al., 1979) and are ATP-dependent (McEntee et al., 1979, 1980). In contrast, efficient annealing can occur also in the absence of ATP (Bryant & Lehman, 1985, 1986) and does not require saturating amounts of RecA protein (Weinstock et al., 1979; Bryant & Lehman. 1985; McEntee, 1985). Tn this paper we reinvestigate the relation between RecA-mediated annealing and recombination. We show that there are two different mechanisms of RecA-mediated ssDNA annealing, which reflect two different steps of the in vitro recombination reaction. One mechanism is related to the preliminary step of homology search where RecA induces homology-independent coaggregation of all available DNA molecules. Within such coaggregates complementary protein-free ssDNA regions can anneal efficiently due to the enhanced DNA concentration. This mechanism alone cannot lead to homologous recognition during recombination reactions involving duplex DNA molecules. However, the enhanced concentrations within coaggregates is important for the stimulation of contacts between protein-free DNA molecules and the stretched and unwound DNA molecules resident in presynaptic RecA-DNA complexes. We demonstrate that these contacts can also lead to annealing. This second mechanism of annealing is related to the actual mechanism of RecA-mediated homologous recognition between protein-free DNA molecules and DNA molecules resident in saturated RecA-DNA complexes.

2. Materials (a) Nucleic

and Methods

acids and proteins

Circular ssDNA molecules with complementary inserts were obtained by cloning fragments of 4X174 DNA into

M13mplO or M13mpll DNA cleaved with Patl and Stul. A 300 bp fragment of 4x174 DNA was produced by exonucleolytic digestion of WuI-linearized 4X174 DNA with BaZ31, followed by restriction with PstI. The ssDNA of phage clones M13c300 and M13nc300 were complementary to each other only in the region of the insert (the remaining parts of the 7.8 kb long DNA molecules were identical). ssDNA was isolated from E&&chia coli strain ,JMlOl infected with phage particles (Messing, 1983). [3H]thymidine-labeled sDNA was obt,ained by growing the phages in M9 minimal medium (Messing, 1983) supplrmented with 0.10; Casamino acids and 10 PCi of [3H]thymidine/ml. Phage dsDNA was isolated by a standard plasmid isolation procedure (Maniatis et al.. 1982). The concentration of DNA was determined spectrophotometrically by using 1 d,,, = 36 pg/ml for ssDNA and 1 AZ6c = 50 pg/ml for dsDNA. DNA concentration and amounts are expressed as concentration of DNA (PM) or amounts of DNA nucleotides (nmol). nucleotides RecA was isolated from the E. coli strain KM4104 transformed with the plasmid pDR1453 (described by Sancar K: Rupp, 1979). RecA was purified by t’he procedure described by Cox et ul. (1981) to fraction IT1 (without ssDNA column). The concentration of RecA was determined using a E2,, 1 “/bof 6.33. according t,o Tsang et al. (1985). Hist’one HI was isolated from rat liver nuclei and purified according t’o the procedure of Sanders dz ,Johns (1974).

(b) Procedures

used to study annealing stimulated by DNA aggregation

Annealing reactions analvzed in Figs I and 2 were in 25 mw-Tr& HCI performed (pH 7.5). I mMdithiothreitol. 40 mM-MgC1,. without or with I mM-ATP (as indicated) at 37°C. DNA c*oncent,ration (in nucleotides) was 12 PM of each partner, and these reactions were start’ed by adding Reed after 5 min of preincubation of the reaction mixt,urr. For t,hc annealing reactions analyzed in Fig. 3. M13c300 ssDNA and AM13nc300 ssDNA were incubated separatei? with RecA in 25 mM-triethanolamine acetate (pH 7.5). 5 m.n-ATP and 1 mM-MgCl, (to avoid aggregate formation). After 5 min, the preformed complexes were mixed. In parallel samples, incubation was either continued at I mM-MgCl, or at 21 miv-MgC1, (to induce aggregate formation). To analyze the DNA produc*ts of the annealing reations. portions were adjusted t,o @5:,,, (w/v) SD8 and 50 rnM-EDTA, analyzed by 0.8?(, (w/v) agarosr gel electrophoresis (prepared in buffer with 1 x TAE 0.5 pg/ml ethidium bromide, according t,o Maniatis et al., 1982) and photographed in ultraviolet light. To quantify the extent of reactions, negatives of the pictures were scanned in a Shimadzu C8 930 TLC:-scanner. The peak areas of product and reactant bands were determined. The extent of annealing was calculated by dividing the peak area of the product band by the sum of the areas of product and reactant bands. The deviation between 2 experiments was found to be about IO9;,. In csontrol reactions. where R,ecA protein was replaced by bovine serum albumin (Sigma) or ovalbumin (Sigma), stimulation of annealing was at least. one order of magnitude lower. The assay to detect RecA I)NA aggregation was performed as described by Tsang rt al. (1985) and consisted of measuring t,he percentage of I3 Hlthvmidine-labeled ssDNA pelleted after 2 min of centrifugation in a micro-

RecA-mediated Annealing of ssDNA fuge (at 12,000 g). Aggregation was also detected by strong gel retardation of glutaraldehyde-fixed networks of RecA-ssDNA complexes. Annealing of ssDNA molecules by thermal treatment and by precipitation with ethanol: for thermal annealing, 12 PM of each partner (M13c300 ssDNA and M13nc300 ssDNA) were incubated for 30 to 60 min in 1 x SSC (150 m&r-Nacj, 15 m&i-sodium citrate) at 56°C and then put to room temperature. For ethanol precipitation, 24 PM of ssDNA (12 PM of each partner) were incubated in 100 m#-sodium acetate, 66% ethanol at -20°C for 4 h. The DNS was pelleted by centrifugation (12,000 g, 10 min) and resuspended at a concentration of 24 PM in 10 mM-Tris. HCl, 1 mM-EDTA (pH 8). (c) Annealing reactions performed with purijed

RecA-DNA

complexes

RecA-ssDNA-ATPyS complexes were formed in 25 m&r-triethanolamine acetate (pH 7.5), 1 mM-dithio threitol, 2 mM-magnesium acetate, 1 mM-ATP at 37°C. ssDNA (30 PM) was incubated with either 1.6, 3.2 or 60 pM of RecA (molar ratios of RecA to nucleotides: 1 : 18, 1 : 9 in 50 ~1. After 5 min, the reaction and 2 : 1, respectively) was adjusted to 2.5 mM-ATPyS (Boehringer) and the incubation continued for 20 min. Excess RecA was then removed by gel filtration on a 1 ml Sepharose 2B column acetate with 25 mivr-triethanolamine equilibrated (pH 7.5), 1 m&r-dithiothreitol. 2 mM-magnesium acetate, 1 mM-ATPyS and eluted with the same buffer in fractions of 100 to 150 ~1 at 37°C. Approximately 1 nmol complexed DNA (in 100 to 150 ~1) was obtained by this procedure and used in the subsequent experiments. The separation of the complexes from unbound RecA protein was confirmed by protein and DNA gel electrophoresis and by electron microscopy. For electron microscopy and nucleoprotein agarose gel electrophoresis, column-purified complexes were fixed with 92% glutaraldehyde at 37°C for 15 min. Electron microscopy was performed as described (Stasiak et al., 1981). resident in $nnealing between ssDNA the RecA-ssDNA-ATPyS complexes and protein-free ssDNA column-purified added to preformed complexes: RecA-ssI>NA-ATPyS complexes (see above) were mixed with protein-free DNA and were incubated further at 37°C. The presence of a band of annealed molecules on gels with samples deproteinized with SDS and EDTA indicated that annealing was occurring during the reaction. The interaction between [3H]thymidine-labeled DNA and unlabeled DNA was detected by fluorography. The gels were soaked for 90 min in En3Hance (New England Nuclear) and then for 90 min in deionized water, dried and exposed to Fuji RX X-ray films. For quantitation, autoradiograms of the gels were scanned in a Shimadzu CS 930 TLC-scanner. To detect the interactions between complexes and protein-free DNA portions of mixtures of RecA-ssDNAATPyS complexes and protein-free DNA were fixed with 0.2O/, glutaraldehyde for 15 min at 37°C and subsequently analyzed by agarose gel electrophoresis. The binding of the added protein-free ssDNA by the preformed complexes resulted in a gel mobility retardation of the bound SSDNA.

3. Results (a) Annealing Using

the

Ml3

promoted by aggregation of ssDNA cloning

a pair of circular ssDNA

system, we have prepared molecules (M13c300 ssDNA

133

and M13nc300 ssDNA) that are complementary to each other only in a 306 bases long region derived from 4X174 DNA (Fig. l(a)). The usage of constructs with such short complementary regions (about 4% of the total length of the circular singlestranded Ml3 DNA), decreases the speed of spontaneous annealing of the two partner DNAs and limits the chance that several ssDNA molecules will anneal to neighboring regions of a single complementary ssDNA molecule. Co-annealing of several ssDNA molecules, giving rise to a complicated pattern of DNA bands on gels, was observed when ssDNA molecules with complementary inserts of 1565 nucleotides and longer were used as a substrate for RecA-mediated annealing (Kenner & McEntee, 1984). Reactions giving just one product band on the gel are more convenient to analyze quantitatively by the gel scanning method. As shown schematically in Figure l(a), the annealing product of ssDNA circles with complementary inserts should have a net linking number of zero (Stettler et al., 1979). The short annealed regions can form a regular right-handed helix while the rest of DNA, consisting of non-complementary regions could form loose left-handed compensatory turns, The micrograph of two annealed ssDNA circles shown in Figure l(c) is consistent with this structure of annealed molecules.

(i) RecA-promoted ssDNA aggregates

annealing occurs within

It has been demonstrated that DNA aggregation facilitates RecA-mediated strand exchange (Tsang et al., 1985). A similar function of DNA aggregation has been proposed for the RecA-mediated DNA annealing reaction (Bryant & Lehman, 1985, 1986; McEntee, 1985). To reinvestigate whether aggregation stimulates annealing we decided to test if RecA-promoted DNA aggregation precedes DNA annealing. Reactions were performed under a set of extremely different conditions and the extents of DNA aggregation (by sedimentation assay) and DNA annealing (by quantitation of DNA bands on gels) were determined. In particular we performed reactions in the presence and absence of ATP knowing that RecA-ssDNA complexes have quite different structures under these conditions (Koller et al., 1983; Stasiak & Egelman, 1986, 1988). We also compared conditions where RecA was provided in amounts sufficient to saturate all ssDNA molecules present in the annealing reaction (saturated RecA-ssDIVA complexes have a stoichiometry of 1 RecA protomer/3 nucleotides; Stasiak & Egelman, 1988), with conditions where ssDNA molecules could be only partially covered with RecA. Time-courses of the RecA-promoted annealing and aggregation were followed at MgCl, concentration of 40 InM (a concentration found to be optimal for several different conditions of annealing). As shown in Figure 2 the strong aggregation (Fig. 2(b) and (d)) was detected at earliest time points under all these conditions while the annealing seemed to

134

B. Mdler

and A. &asiak

(a) Reactants

(R)

Product

(P)

(b)

Cc)

Figure 1. Appearance of annealing products on agarose gels and in the el&ron microscope. (a) Schematic, drawing of the reactant (R) ssDNA molecules with complementary inserts and the product) of the annealing reaction (I’) (complementary and non-complementary regions of the ssDNA molecules are drawn out of proportion). (b) &arose gel showing the analysis of DNA reactants and products of the annealing rea,ction. Lanes I. 2. 3. the thermal annealing procedure performed with M13nc300 ssDNA only, with M13nc300 ssDXA only and with an quimolar mixturr ot M13c300 SSDNA and M13nc300 ssDNA. respectively. Lane 4. a mixture of 2 ssDN;A partners was preincuhatrd in 25 mM-Tris HCI (pH 7.5), 1 mM-dithiothreitol. 40 mM-MgCl,, 1 miv-ATP and stimulated to anneal by the subsequent addition of RecA and incubated for 15 min. Lane 5, the same conditions as for the reacstion in lane 4. but RecA was replaced by bovine serum albumin. The reactions were stopped and the products were analyzed as dewribed in Materials and Methods. (c) Electron micrographs of reactant (R) and products (P) of the annealing reaction. Hgpophase spreatling of DNA was performed according to Sogo et al. (1979) wit’h 309, formamide. Formamide melt,s srcondarv regions in ssDNA and premelts duplex DNA. This leads to a substantial decrease of DXA helicity in the paired region (stretc,h between the 2 arrows) and explains why we did not, observe 30 wrappings compensating for the formation of 300 bp of duplex DNA. In the schematic drawing t,he 2 reactant, strands are drawn to different thickness. The length of the magnification bar represents 170 nm.

RecA-mediated

Annealing

qf ssDNA

135

(b)

i.i:“‘-

-+-O-

0

10

1pMRec 24 fl

Ret 90

30

;0

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10 20 30 Time of incubation (min)

90

0

10

90

Time of incubation (min) (d) No ATP ‘O”-

-l

“” 0

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Time of incubation (min)

Time of Incubation (mln)

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2.5

5

10

15

20

30

90

1

2.5

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Time (min)

Time (min)

Figure 2. Time-courses of RecA-mediated ssDNA annealing and aggregation. RecA-mediated annealing reactions between M13c300 ssDNA (12 PM) and M13nc300 ssDNA (12 PM) were performed with 1 pM or 24 q-Red protein (molar ratio of RecA to nucleotides of DNA 1 : 24 or 1 : 1, respectively) as described in Materials and Methods. The reactions were performed either in the presence of 1 mM-ATP ((a), (b), (e), (0) or in the absence of ATP ((c) and (d)). To quantify the extent of the annealing reaction, negatives of the photographs of the gels (like those shown in (e) and (f)) were scanned and the fraction of DNA in the product band was determined. The extent of DNA aggregation was determined by a centrifugation assay as described in Materials and Methods. (a) and (c) Time-courses of annealing in the presence and absence of ATP, respectively. (b) and (d) DNA aggregation in reactions in the presence and absence of ATP, respectively. (e) and (f) Agarose gels showing the time-courses of annealing performed in the presence of ATP by RecA provided in undersaturating (e) and saturating amounts of RecA protein (f). These 2 gels were used for the data points displayed in the graph (a). The position of the reactant and the product band are marked R and P, respectively.

follow

the

Interestingly

aggregation the

aggregates

(Fig.

Z(a)

and

(c)).

dispersed

with

time,

the annealed molecules remained paired. The ATPase activity of RecA causes accumulation of ADP in the reaction mixture (Cox et al., 1983; Menetski & Kowalczykowski, 1985) leading to the dissociation of RecA-ssDNA complexes. This dissociation can in turn result in the dispersion of the aggregates in the reactions supplemented with ATP. while

In the reactions performed without ATP the partial dispersion of aggregates might by due to a competition for RecA between RecA-ssDNA complexes and RecA microcrystals. RecA microcrystals are known to form easily under elevated MgCl, concentrations 1987). The dissociation of (Griffith & Harris, RecA-ssDNA complexes indicated that even in the presence of saturating amounts of RecA, increasing amounts of protein-free ssDNA stretches appeared

136

B. Miiller and A. Sta.siak

in the aggregates. Therefore the lag phase of annealing observed in the reaction performed in the presence of ATP and saturating amounts of RecA (Fig. 2(a) and (f)), could indicate the dependence of annealing on the presence of protein-free ssDNA stretches in the aggregates. DNase accessibility studies (data not shown) were consistent with the progressive appearance of protein-free ssDNA regions in these types of aggregates. In mock annealing reactions containing only one type of partner single-stranded DNA molecules, aggregation occurred to the same extent as in the real annealing reactions (data not shown). This indicates that the aggregation is indeed independent) of annealing and can therefore precede annealing. The design of the experiments presented in Figure 2 does not allow us to conclude directly whether RecA-saturation of single-stranded DNA could inhibit annealing. Since the reaction was started by

satur.

unsatur. SDS

the addition of RecA to premixed ssDNA partners. the annealing could start before RecA-covering of the partner DNA molecules has been completed. The reactions analyzed in Figure 3 avoid this draw back. Tn these reactions, the partner DNA molecules were separately complexed with RecA and only then mixed for annealing. (ii) Coaggregation and the prrsencr qf protein-frur ssDNA stretches in coaggregates are required xnDNA annealing

fbr

To firmly establish a causal link hetween the RecA-mediated ssDXA aggregation and annealing. we decided to follow the approach of Tsang et al. (1985), who demonstrated that DNA aggregation is the an instrumental, preliminary of step RecA-mediated in vitro recombination reaction. in particular they demonstrated that for efficient

satur.

unsotur. glutaraldehyde

Figure 3. The formation of coaggregates is necessary for stimulation of annealing. Ml3c300 SSDNA (11 PM) arid M13nc300 ssDNA (12 PM) were incubated separately with RecA for 2.5 min in 25 m.n-Tris HCI (pH 7.5). I mM-MgCl,, 5 rnzvl-ATP. and only then mixed together. Preincubations were performed with 6 ,aM-RecA. to form saturated complexes (using 1 RecA/2 nucleotides) or with 1 PM-RecA. to form unsaturated complexes (using 1 RevA/ nucleotides). The low magnesium concentration allows for the formation of RecA-ssDNA complexes but does not induce aggregation of the complexes. The preformed complexes were mixed together according to 3 different prot,ocols: (1) mixed only (m). the mixture was incubated unchanged. without addition of MgCl, (lanes 1 and 7 for saturated and lanes 4 and IO for unsaturated complexes); (2) mixed and aggregated (m and a). after mixing the M&I, concentration was increased to 21 mM to induce aggregation (lanes 2 and 8 for saturated and lanes 5 and 1 I for unsaturated complexes); (3): aggregated and mixed (a and m). the complexes were adjusted separately after 2.5 min to 21 mrvr-MgCf, t’o induce self-aggregation. After a further 2.5 min, the complexes were mixed together and the incubation continued (lanes 3 and 9 for saturated and lanes 6 and 12 for unsaturated complexes). After 5 min of coincubation, all reactions were stopped by addition of SDS to analyze the DNA products of the reaction (lanes 1 to 6) or by addition of 0.1 96 glutaraldehyde to analyzr nucleoprotein complexes (lanes 7 to 12). Marker lanes M M13c300 ssDNA.

RecA-mediated Annealing recombination reaction the partner DNA molecules should be mixed before the RecA-induced aggregation, to allow the formation of interspersed coaggregates. The enhanced DNA concentration within the coaggregates stimulated the frequent intermolecular contacts between the partner DNA molecules, promoting the search for homology. When the partner DNA molecules were aggregated separately and then mixed, they were unable to form interspersed coaggregates and therefore unable to interact with each other. To test whether ssDNA annealing in aggregates occurs between RecA-free or RecA-covered DNA regions we compared t’he ethciency of annealing in aggregates formed by unsaturated and saturated RecA-ssDNA complexes (input ratios of 1 RecA/lS or 2 nucleotides, respectively). Since we decided to work with the complexes formed in the presence of ATP, the time of coincubation of the preformed complexes was shortened to five minutes and the ATP concentration was increased to 5 mM to minimize the destabilizing effects of the accumulation of ADP. RecA complexes were formed separately with each of the two annealing partners, under conditions does not occur where aggregation (1 mM-MgCl,). Then the complexes formed with the two annealing partners were mixed together under three different conditions: (1) complexes were mixed at 1 mM-MgCl, (without formation of aggregates); (2) complexes were mixed and aggregate formation was induced by raising the MgCl, concentration; and (3) complexes were aggregated separately by raising the MgCl, concentration, then mixed and further incubated. The gel shown in Figure 3 shows the result of such an experiment. SDS-treated samples (Fig. 3, lanes 1 to 6) reveal that significant annealing occurred only in the reaction where unsaturated complexes formed with both annealing partners were mixed before the formation of aggregates (Fig. 3, lane 5). If mixing was done after the aggregate formation or if the mixed samples were not induced to form aggregates there was no significant annealing (Fig. 3, lanes 6 and 4, respectively). Glutaraldehyde-fixed samples (Fig. 3, lanes 7 to 12) reveal that the rise of the MgCl, concentration was equally efficient in the stimulation of aggregate formation in the case of unsaturated and saturated RecA-ssDNA complexes (compare Fig. 3, lanes 89 with lanes 11-12). However, aggregation of saturated complexes did not promote significant annealing (Fig. 3, lanes 2 and 3). Notice that at a MgCl, concentration of 1 mM, the fixed RecA-ssDNA complexes can enter agarose gel although the saturated the 1% complexes are strongly retarded in their migration (Fig. 3, lane 7). However, after increasing the MgCl, concentration to 21 mM, the glutaraldehyde-fixed complexes cannot enter the gel, indicating the formation of large intermolecular networks. These findings demonstrate that protein-free ssDNA stretches on both partner DNA molecules as well as RecA-mediated coaggregation of the partner ssDNA molecules are required for efficient annealing.

qf ssDNA

137

(iii) Stimulation of annealing by aggregation is not a

unique property of proteins that recognize homology between interacting DNA molecules The stimulation of ssDNA annealing described above occurred in the presence of RecA. This might give the impression that the specific ability of RecA to recognize homology or complementarity between two interacting DNA molecules plays an important role in these annealing reactions. However, if just the property of RecA to aggregate DNA and not its ability to recognize homologous or complementary DNA strands is important for the efficient stimulation of the annealing reactions, then also other ssDNA aggregating agents should be able to stimulate annealing. To test this possibility, we tried to perform annealing reactions in the absence of RecA, but under conditions that promote ssDNA aggregation. It is established that histones induce annealing of ssDNA and histone-induced DNA aggregation was proposed to be responsible for increasing the local concentration of ssDNA, promoting thus efficient annealing (Cox & Lehman, 1981). The gel in Figure 4(a) shows a time-course of histone Hl-promoted annealing. The reaction was very fast, already after 20 seconds more than 80% of the DNA was annealed. As expected, the efficient annealing mediated by histone Hl was associated with the aggregation of the ssDNA molecules (Fig. 4(b)). In the presence of histone Hl several DNA product bands were formed (Fig. 4(a)), in contrast to the reactions performed with RecA, where mainly one product was observed (see Figs l(b) and 2(e) and (f)). This can be explained by the differences in the speed of the two reactions. In the presence of histone Hl the annealing reaction seems to have a higher initiation rate, leading to several nucleation events within the individual 300 bp long stretches of complementarity. These multiple initiations can lead to the formation of composite annealed molecules (slower migrating additional product bands in Fig. 4(a)). RecA-stimulated annealing between partially complementary circular ssDNA molecules can also give rise to composite annealed DNA molecules (Kenner & McEntee, 1984). However, this requires much longer complementary sequences than those used in this work (data not shown). Bryant et al. (1989) reported that simple annealing products appear before the composite annealing products but it is likely that such phenomena do require the existence of long complementary sequences. Since ssDNA annealing could be one of the biological functions of histone Hl, we turned to protein-independent methods of ssDNA aggregation like ssDNA precipitation by ethanol. As predicted, this method of aggregation also promoted efficient ssDNA annealing (Fig. 4(c)). The finding that both histone Hl and precipitation with ethanol promote annealing (while such procedures are not known to stimulate recombination reactions) is consistent with the idea that the

138

13. Miiller and A. Stasiak

-R P

R

-HI

20”

2-5’

40”

5’

IO’

30’

(a)

1

1

I

5

I

I

_

so

IO Time (mm) (b)

Figure 4. Aggregation and annealing of ssDNA by histone HI or by precipitation with ethanol. (a) 11113~300 ssl)SA (12 PM) and M13nc300 ssDNA (12 ,UM) were incubated in 25 mM-Tris . HCI (pH 7.5). 10 mM-Mgci, at 37°C for 5 min. Then histone Hl was added to a concentration of 8 pg/ml. At the indicated times, portions of the reaction were stopped by adding SDS and EDTA to 1 y0 (w/v) and 50 mM, respectively. and subsequently analyzed by agarose gel electrophoresis. The position of ssDNA (R). of the major annealing product (P) and of minor annealing products composed presumably of more than 2 ssDNA molecules are indicated (arrows). (b) Proportion of ssDNA in aggregates (determined as described in Materials and Methods) at different’time points of incubation with histone H 1. (c) Producats of ssDNA

annealing

by precipitat,ion

with ethanol.

The assignment

ability of RecA to aggregate ssDNA and not its pairing activity is the main driving force for the ssDNA annealing in the RecA-ssDNA aggregates. (b) Annealing molecule

promoted by the uptalce of a ssDNA into RecA-ssDNA complexes

In this section we study annealing reactions where one partner strand is completely covered with RecA and the complementary strand is protein-free. It’ has been demonstrated that during the presynaptic stage of the in vitro recombination reaction, ssDNA is complexed with RecA while dsDNA remains protein-free (Stasiak et al., 1984; Stasiak & Egelman, 1986: Register et al.. 1987). The reaction

of the bands

is as for (a).

between saturated RecA-ssDNA complexes and dsDNA takes place by a progressive uptake of the protein-free homologous dsDNA into the RecA-ssDNA complex (Stasiak & Egelman. 1988; Miiller et al., 1990). We have shown that direct DNA- DNA contacts are possible between the ssDNA resident in the complex and protein-fret, ssDNA and that these contacts can lead to annealing (Miiller et al., 1990). Here we characterize further this type of RecA-mediat,ed annealing. (i) Saturation of RecA-ssDNA con~pleze~s is required for their reactivity with protein-free ssDNA To test for int,eractions between ssDNA molecules and protein-free

RecA-covered ssDNA thtx

R&CA-mediated Annealing of ssDNA preformed complexes were stabilized by the addition of ATPyS to the complexes formed in the presence of ATP. Otherwise, RecA protomers would redistribute quickly between preformed complexes and subsequently added protein-free ssDNA (Menetski & Kowalczykowski, 1985; Neuendorf & 1986). In the presence of ATPyS COX, RecA-promoted branch migration does not traverse such long stretches as in the reactions performed in the presence of ATP (Menetski et al., 1990; Rosselli & Stasiak, 1990). However, the RecA-ssDNA complexes formed in the presence of ATPyS (RecAssDNA-ATPyS complexes) are fully active in

M

I

3

2

4

5

139

binding homologous duplex DNA and promote the reaction of the recombination initial phases including the process of homologous recognition (Honigberg et al., 1985; Miiller et al.,1990). To be active in the recombination reaction with dsDNA, RecA-ssDNA complexes must be saturated with RecA protein (Shibata et al., 1979). We have shown that binding and annealing of singlestranded oligonucleotides to single-stranded DNA resident in the RecA-ssDNA-ATPyS complexes depends on RecA saturation of these complexes (Miiller et al., 1990). Here we decided to test whether these rules apply also for interactions of long ssDNA

7

6

8

M

9

P

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I:9 t

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+

2:i -

+

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DNA

Glutaraldchyde

Figure 5. Saturation of RecA-ssDNA-ATPyS complexes with RecA protein is required for annealing with protein-free ssDNA. RecA-ssDNA-ATPyS complexes containing M13c300 ssDNA and varying amounts of RecA protein were formed and purified as described in Materials and Methods (complexes formed with molar ratios of RecA to nucleotides: 1 : 18. 1 : 9 and 2 : 1 are shown in the indicated lanes). Portions (05 nmol) of purified complexes were either fixed with glutaraldehyde for analysis by electron microscopy (Fig. 6) and by agarose gel electrophoresis (lanes 4, 6 and 8), or were supplemented with 4X174 (+) ssDNA (@6 nmol) and further incubated for 15 min at 37°C. These incubations were stopped by the addition of SDS and EDTA (lanes 1, 2 and 3) or glutaraldehyde (lanes 5, 7 and 9) prior to the analysis by agarose gel electrophoresis, as described in Materials and Methods. Marker lanes (M) contain $X174 ssDNA, M13c300 ssl)NA and the product position of unsaturated

of thermal annealing RecA-ssDNA-ATPyS

with these 2 ssDNA complexes.

molecules

(P). The arrows in lanes 4 and 6 indicate

the

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molecules with preformed RecA-ssDNA-ATPyS complexes. RecA-ssDNA-ATPyS complexes were formed with M13c300 ssDNA and either non-saturating (RecA to nucleotide ratios of 1 : 18 and 1 : 9) or saturating amounts of RecA (RecA to nucleotide complex formation, the ratio of 2 : 1). After unbound RecA was removed by gel filtration: The complexes were then mixed with protein-free, partially complementary 4X174 ssDNA molecules (containing 300 nucleotides of complementary sequences). To avoid aggregation, the reactions were performed in 2 mM-MgCl,. These conditions are suitable for homologous recognition in recombination reactions performed in the presence of ATPyS (Honigberg et al., 1985; Rosselli & Stasiak, 1990). After deproteinization the two types of reactant’ molecules and the anealed molecules migrate each with different mobilities (see Fig. 5, marker lanes). The interaction between complexed and protein-free ssDNA was tested for the appearance of annealing products after deproteinization of DNA (Fig. 5, lanes 1 i 2 and 3). In addition, portions were fixed with glutaraldehyde for the analysis of the complexes and the products of their interaction with ssDNA (Fig. 5, lanes 4 to 9) by agarose gel electrophoresis. This method allows direct detection of the interaction between protein-free DNA and the preformed complexes and is not dependent on the formatsion of annealing products. Glutaraldehyde-fixed complexes formed at the molar ratio of RecA to nucleotides of 1 : 18 were visible on the grl as a rather fast migrating smear, indicating that these complexes had a relatively small molecular weight and were heterogeneous in size (Fig. 5. lane 4, region between the 2 arrowheads). Complexes formed at the RecA to DNA ratio of 1 : 9 migrated much slower but still in the form of a smear (Fig. 5, lane 6, region between the 2 arrowheads). In contrast, complexes formed with saturating amount,s of RecA (RecA to DNA ratio of 2 : 1) migrat,ed as a highly retarded sharp band (Fig. 5, lane 8). The appearance in the electron of t’hese RecA-ssDNA-ATPyS microscope complexes (Fig. 6) was in good agreement with their mobility during gel electrophoresis (Fig. 5). This demonstrated that, with increasing RecA to ssDNA ratio, the complexes became more extended and more homogeneous in size. It was visible t’hat only the saturated complexes (Fig. 6(c)) had the characteristic structure of regularly striated and fully extended presynapt,ic RecA-ssD?C’X complexes (Flory ef nl.. 1984; Koller it nl., 1983: Heuser & Criffit,h, 1989). Only saturated RecA-ssDsA-ATPyS complexes had the ability to interact, wit,h added protein-free DNA. The undersaturated complexes shown in Figure 5, lanes 5 and 7, did not interact with the added naked ssDNA, since the $X174 ssDNA appeared as a sharp band on the gel at the position of protein-free 4x174 ssDNA. In contrast. in the reaction with saturated complexes (Fig. 5. lane 9). added protein-free 4x174 ssDNA was not visible.

(b)

I 100 nm

Figure 6. Electron micrographs ot’non-saturated and saturated Rec&ssD~LATPyS caomplexes. EleCtron micrographs (a). (b) and (c) show complexes analyzed on lanes 4. 6 and 8 in Fig. 8. respectively. Glutaraldehydefixed complexes were adsorbed on glow-discharged carbon supports from a, 2 rnx-magnesium acetate solution and. after dehydration. rotary shadowed with platinum/carbon (Stasiak rt al.. 1981). hut all the fluorescence could be seen in the slot This suggests that saturated complexes bind the added 4X174 ssl)NA: this leads to the formation of aggregates that cannot enter the gel. Aggregate formation is individual expected as the RecA-ssDNA-ATPyS complexes can start t,o

RecA-mediated

Annealing

interact with several protein-free DNA molecules of which every one can be attached to several preformed complexes. This polyvalency of interaction between RecA-DNA complexes and proteinfree DNA molecules leads then to aggregate formation. The deproteinized samples (Fig. 5, lanes 1, 2 and 3) showed that only the interaction of saturated complexes with 4X174 ssDNA (lane 3, weak band at the height of P band of the marker lines) lead to an annealing product. This demonstrates that the saturation of ssDNA with RecA is required for the ability of the complexes to bind protein-free ssDNA and to promote the annealing between the strand resident in the complex and the strand additionally bound by the complex. (ii) Complementarity-independent stable associations between preformed, saturated RecA-ssDNAATPyS complexes and coincubated protein-free ssDNA limit the extent of annealing During RecA-mediated presynaptic RecA-ssDNA

MI

in vitro recombination the complexes stably bind

2345676M

-P

(a)

-R

homologous duplex DNA while the interaction with non-homologous duplex DNA is only of transient nature (Honigberg et al., 1985; Miiller et al., 1990). This indicates that the homology recognition process requires non-homologous contacts to be short lived. This would allow for new contacts occurring many times until the homologous contact, which leads to a more stable interaction, takes place. The observation that preformed, saturated RecAssDNA-ATPyS complexes stably bind both complementary and non-complementary protein-free ssDNA (Miiller et al., 1990) indicates that the complementarity dependence and the selectivity of this process can be low. We therefore decided to quantify more accurately the efficiency and the timecourse of the reaction between ssDNA in saturted RecA-ssDNA-ATPyS complexes and protein-free partially complementary ssDNA using radioactively labeled ssDNA. After the indicated times of incubation, portions of the reaction were deproteinized, analyzed by agarose gel electrophoresis and the DNA visualized by fluorography. Figure 7(a) and (b) shows that within five minutes the reaction reached a plateau value with about 8% of the ssDNA molecules annealed. The rapid kinetics of this annealing reaction and the relatively low efficiency are consistent with the observation of a rather non-selective (complementarity-independent) binding of protein-free ssDNA by saturated RecA-ssDNA-ATPyS complexes.

4.

0*3’0.6’ I ’ 2.5’ 5’ IO’20’ (a) Annealing

’ 1 ’ 1 1 1 I 2.5 5.0 7.5 100 12.515.0 17.5 20.0 Time of incubation

(min)

Figure 7. Annealing between saturated RecA-ssDNA-ATPyS complexes and partially complementary ssDNA is inefficient. RecA-ssDNA-ATPyS complexes containing [3H]thymidine-labeled M13nc300 ssDKA were formed (at a molar ratio of RecA to nucleotide of 2 : 1) and subsequently purified as described in Materials and Methods. Annealing was started by adding M13c300 ssDNA (66 nmol) to the complexes (1 nmol). At the times indicated portions of the annealing reaction were stopped by addition of SDS and EDTA, analyzed by

agarose gel electrophoresis and visualized by fluorography. (a) Fluorogram. Lane 1, complexed ssDNA before the addition time-course produced by ssDNA. R, Quantitation as shown in

of the complementary ssDNA. Lanes 2 to 8 of annealing. The marker lanes M were thermal annealing of M13c300 and M13nc300 ssDNA and P, annealing product. (b) of a less exposed fluorogram of the same gel (a).

141

of 8sDNA

Discussion

stimulated by DNA

aggregation

We systematically explored the reaction conditions that are suitable for the RecA-mediated annealing of ssDNA and noticed that RecA-induced ssDNA aggregation seemed to precede the actual annealing reaction (Fig. 2). RecA-induced DNA aggregation was efficient also in the absence of complementary ssDNA, demonstrating that aggregation is independent of annealing. The observation that only the formation of interspersed coaggregates composed of unsaturated RecA-ssDNA complexes lead to efficient annealing indicates that annealing occurs between protein-free ssDNA regions of coaggreagated annealing partners (Fig. 3). It was shown earlier that ssDNA molecules resident in the RecA-ssDNA-ATP or -ATPyS complexes are located close to the axis of the complexes with a diameter of 100 A (1 A = 0.1 nm) (Egelman & Yu, 1989). Therefore, even if these complexes align side-by-side (Egelman & Stasiak, 1986, 1988) ssDNA molecules from different. saturated complexes are unable to contact each other. This explains why aggregates composed of fully saturated complexes did not stimulate annealing (Fig. 3). Bryant & Lehman (1985) came earlier to similar conclusions. An analogous situation was also reported for the complexes formed between DNA and UvsX protein. UvsX, the recombination promoting protein of phage T4, is unable to mediate

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ssDNA-dsDNA recognition when each of the partner DNA molecules is complexed with LJvsX protein. However, ssDNA-dsDNA recombination can take place when only ssDNA is complexed with UvsX protein (Harris & Griffith, 1987). The data presented in Results. section (a), suggest that the main factor responsible for the RecA-stimulated ssDNA annealing is ssDNA aggregation, which promotes frequent contacts between molecules. To interacting ssDNA stimulate annealing it is not enough to increase the local concentration of interacting ssDNA molecules. Tn addition, it is necessary that the ssDNA molecules are free to contact’ each other and that the residues involved in complementary hydrogen-bonding are not occupied by the aggregat’ing agent. In fact, it is relatively easy to form such aggregates and it does not require t’he presence of a protein t,hat could recognize complementaritv of ssDNA molecules. As shown in Figure 4(c), a simple ssI>NA aggregating procedure such as precipitation wit’h ethanol stimulated ssDNA annealing to a higher extent than RecA did. The well known efficient) annealing reaction mediated by RecA seems not to reflect the specific DNA aligning activity required during the RecA-mediated recombination reaction. Our results indicate that’ it only reflects t,he abilit,y of RecA to aggregate DNA. This aggregation is, however, an important preliminary step for t’he actual pairing st’age of t)he recombination reaction. Tn these reacincreases the frrquency of tions aggregation contacts of crotein-free DNA molecules with DNA molecules exposed for pairing in saturated presynaptic RecA-DNA complexes (Tsang Pt ~1.. 1985). (b) il nnealiny occurriny between protein,-frPP ssIjLVA and cssDNA resident in thr satu,ratrd IZe(:A~ssDX~TA cwmplezes Mixing RecA-ssDNA_ATPyS complexes formed under non-saturating and saturating conditions with partially complementary prot,ein-free ssDNA showed that only complexes saturated with RecA protein interacted with protein-free ssDNA (Fig. 5). This is caonsistent with the known behavior of RecA-ssDNA complexes during the in &ro recombination reaction (Shibat,a ct al.. 1979). Under saturating conditions. ItecA bound t,o ssDNA forms a deeply grooved helical filament with a pitch of 95 A (Egelman &, Stasiak, 1986; Stasiak & Egelman. 1988: Heuser & Griffith. 1989). This filament serves the function of a recombinational scaffold (Griffith 8 Formosa, 1985). The ssDNA molecules contained wit’hin this scaffold adopt a stretched configuration with all bases prepared for the process of DNA-DNA rrcogniGon (HowardFlanders rt al., 1984: IWapua 8 Miiller. 1987). complemrntar? protein-free Indeed. partially added to saturated ssDNA molecules RecA-ssI)NA-ATPyS complexes were able to anneal with the strands contained in the preformed complexes (Fig. 5). The occurrenpc of ssDNA annealing indicated that the two l>NA strands could contact each other.

In reactions between RecA-ssI)NA-AT t’@ complexes and protein-free dsDNA. stable interactions are only formed with homologous dsDNA (Honigberg et aZ.. 1985: Miiller d al.. 1990). In contrast. the RecA-ssI>NA-ATPyS complexes do not discriminate between complementary and noticomplementary ssDNA and interact’ with both (Takahashi et al.. 1989: Miiller Pt al.. 1990). This suggests that the low efficiency of annealing undrr these conditions is due to trapping of protein-free ssDNA by the c~omplrxes out of homologous register. This prevents the interacting strands from repeated caont’acts, which would lead eventually to efficient annealing. However, the annealing efficiency (SO&) is higher than would be expected from a reaction where all the tirst contacts are irreversible, suggesting that thrrr might bc a slight disbetween complementary and noncrimination complementary strands. It is probable that when the two strands contact) each other along t,he helical groove of the preformed c*omplexrs. the hydropcknbond formation between the added strand and th(l resident strand contributes tn t’he stabilization of’ this interact,ion. The formation of hydrogen bonds c*ould he decisive in changing initial cont,acLts illt o stable contacts leading to a further envelopment of flanking protein-free ssDSA int)o the c~omplrsc~s. \l’r propose t,hat this further envelopment don not depr~nd on complementjarity between the flanking within thci ssf)NA and t’he ssDxA resident ~mplrxc~s. Thtt observat,ion that KrcA~~ssl)S.-1 ATPyS complexes recognize more selectively homelogous duplexes than complementary ssl)S;! might br explained by a bigger flexibility of ssl1S.4 leading to an induced tit phenomenon. Ac*c~ordinply. protein-fi,ee ssl)NA would be able to adapt its Iocxl structure (by bulging out st ret,cshing or (aomprrssion) to maximize the possibility of hydrogen bonding with the strand residing in thr c*ornplr~rs. This could If&ad to a sufficient densit\. of hvdropt>rl bonds to stahilizt, somt’ of thr non-c.;,rrll,lement~~t,~. interactions. In addition. thr ability of Rw.~ to discriminate hetlvren c,oml)lernnltwr,?and t1011bta artificGall>. strands might (~(1rnl)lrmt,ntar? suppressed in our experiments bv using thcl no11‘iTI’YS. K~W.4 XTP-analog physiological )1101Y arcs much SSl)?r;A~~&4’I’F’~~S c*omplrxrs stable than RecA~ssDNA~ATP c~omplexrs. t herchfore protein-free ssI)?r;A entering the prt+‘ormt~ti RecA~ssDNA~ATPyS c+omplexcas might bc morn avidly trapped inside of t,hese complexes. I’rrhaps. ptlysiological RevA---ssl)NA-ATT’ c*ornples~~s ret ain the entering ss1)N.A only if the hydrogen bonding between t)he interacting 1)N.A molec~ules is almost c~omplrtr. (C) Krldion brtwern Rev,1 -mediated I)SA utrblbralin(y nnd thp mechanism of hcymo1oyou.s rpcoynition We have shown that RecA can mediattl ssl)NX annealing by a mechanism similar to homologous recognition during recombination. Does this mean that Rec,4 uses the possibility to form Wat,son-(Irick

RecA-mediated

Annealing

143

of ssDNA

strands connected by MeGavin’s hydrogen bonds should spontaneously into regular switch Watson-Crick base-pairing, similar to the mechanism for RecA-mediated recombination proposed by Howard-Flanders and colleagues (Howard-Flanders et al., 1984). We thank Professor Theo Koller for discussions and suggestions leading to the development of this work; Professor Jacques Dubochet, Professor Edward Egelman, Dr Bernadette Connolly, Dr Stephen C. West and Walter Rosselli for helpful discussions and for critical reading of the manuscript; Heidi Meyer-Rosa and Corinne Cottier for help in preparing the Figures. This work was partially supported by Swiss National Fund grants 31-25694.88 and 31-27146.89 (to J. Dubochet and A.%).

References

Figure 8. Two possible mechanisms for RecA-mediated recognition of complementarity between ssDNA molecules. Schematic representation of 2 alternative models for the recognition of complementarity between ssDNA in saturated RecA-ssDNA complexes and protein-free ssDNA. The drawing shows cross-sections of RecA-DNA filaments interacting with complementary ssDNA. Depending on the relative position of the binding sites for the resident strand (site I) and the incoming strand (site II) the interaction between ssDNA molecules could proceed in 2 possible ways. A, The recognition of comple-

mentarity occurs by direct formation of Watson-Crick hydrogen bonding between the resident and the incoming ssDPiA. R, The recognition of complementarity occurs by formation of McGavin’s hydrogen bonds. A subsequent’ rotation of the bases leads then to the formation of Watson-Crick base-pairs.

base-pairs for the homologous recognition between ssDNA and dsDNA or between two dsDNA molecules during recombination reactions? Do our results support the mechanism “strand separation before pairing” and are they against the mechanism “pairing before strand separation” (see Introduction)? Not necessarily, it is possible that the ssDNA resident in the RecA-ssDNA-ATPyS complexes offers only a possibility for the formation of McGavin’s hydrogen bonds with the interacting complementary strand (McGavin, 1977). As drawn schematically in Figure 8, these hydrogen bonds could provide an alternative basis for recognition of complementarity. Two complementary ssDNA

Bryant, F. R. & Lehman, I. R. (1985). On the mechanism of renaturation of complementary DNA strands by coli. Proc. Nat. Acad. the RecA protein of Escherichia Sci., U.S.A. 82, 297-301. Bryant, F. R. & Lehman, I. R. (1986). ATP-independent renaturation of complementary DNA strands by the mutant RecAl protein of Escherichia coli. J. Biol. Chem. 261, 12988-12993. Bryant, F. R.. Menge, K. L. & Nguyen, T. T. (1989). Kinetic modelling of the RecA protein promoted renaturation of complementary DNA strands. Biochemistry, 28, 1062%1069. Cox, M. M. & Lehman, I. R. (1967). Enzymes of general recombination. Annu. Rev. Biochem. 56. 229-262. Cox, IK M. & Lehman, I. R. (1981). Renaturation of DNA: a novel reaction of histones. Nucl. Acids Res. 9, 389-400. Cox, M. M., McEntee. K. & Lehman, I. R. (1981). A simple and rapid procedure for the large scale purification of the RecA protein of Escherichia coli. J. Biol. Chem. 256, 467-678. Cox, M. M., Soltis, D. A., Lehman, I. R., DeBrosse. Ch. & Benkovic, S. J. (1983). ADP-mediated dissociation of stable complexes of RecA protein and single-stranded DNA. J. Biol. Chem. 258, 2586-2592. Cunningham, R. P., Shibata. T., DasGupta, & Radding. C. M. (1979). Homologous pairing in genetic recombination: Single strands induce RecA protein to unwind duplex DNA. Nature (London,), 281. 191-195. DiCapua, E. & Miiller, B. (1987). The accessibility of DNA to dimethylsulfate in complexes with RecA protein. EMBO J. 6. 2493-2498. Egelman, E. & Stasiak, A. (1986). The structure of helical RecA-DNA complexes: I. Complexes formed in the presence of ATP-gamma-S or ATP. J. MoZ. Biol. 191. 677-697 Egelman. E. & Stasiak, A. (1988). Structure of helical RecA-DNA complexes: II. Deformations of RecA-ATP-gamma-S filaments in bundles and the implications for the RecA strand exchange reaction. .I. Mol. Biol. 200, 329-349. Egelman. E. H. & Yu, X. (1989). The location of DNA in RecA-DNA helical filaments. Science, 245, 404407. Flory, J.. Tsang, S. & Muniyappa. K. (1984). Isolation and visualization of active presynaptic filaments of RecA protein and single-stranded DNA. Proc. Nat. Acad. Sci., C'.S.A 81, 702&7030. Griffith, J. D. Formosa. T. (1985). The UvsX protein of

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bacteriophage T4 arranges single-stranded and double-stranded DNA into similar helical nucleoprotein filaments. J. Biol. Chem. 260, 4484-4491. Griffith, J. D. & Harris. I,. D. (1988). DNA strand exchanges. CRC Crit. Rev. Biochem. 23S S43%S86. Harris, 1,. D. & Griffith, tJ. (1987). Visualization of the homologous pairing of DNA catalyzed by the bacteriophage T4 UvsX protein. J. Biol.