atp-dependent unwinding of the double helix and extensive

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 258, No. 20, Issue of October 25, pp. 12394-12404, 1983 Printed in U.S. A .

ATP-dependent Unwinding of the Double Helix and Extensive Supercoiling by Escherichia coli recA Protein in the Presence of Topoisomerase*

(Received for publication, March 23, 1983)

Masa-aki IwabuchiS, Takehiko Shibatat, Takuzo OhtanilT, Masahiko NatoriII , and Tadahiko Ando From the Department of Microbiobty, Riken Institute (The Institute of Physical and Chemical Research), Wako-

”

shi, Saitama,-351, Japan

recA protein, which is essential for genetic recombination in Escherichia coli, causes extensive unwinding of the double helix by an ATP-dependent reaction and accumulation of positive supercoiling in closed circular double-stranded DNA. Initiation of the extensive unwinding was largely dependent on homologous single-stranded DNA. Therefore, it is likely that the extensive unwinding is initiated mainly at the site of D-loops. “Nascent D-loops” in which the two DNA molecules did not interwind were also good initiation sites of extensive unwinding. When the concentration of Mg2+ was decreased from the standard conditions for D-loop formation (13 mM MgC12; the higher Mg2+ condition) to the lower Mg2+ condition (1 to 2 mM MgC12), extensive unwinding by recA protein was initiated very quickly in the absence of single-stranded DNA. Results showed that this single-stranded DNA-independent initiation of extensive unwinding (i) requires negative superhelicity of the double-stranded DNA and (ii) is a first order reaction with respect to the DNA. These observations suggest that, under the lower Mg2+ condition, the extensive unwinding starts at a tram- siently denatured site in the negative superhelical DNA. Once initiated, the unwinding by recA protein is propagated extensively, even under conditions that do not allow its initiation. Therefore, the propagation of unwinding is a processive reaction (“processive unwinding”). Previous studies indicated that recA protein promotes “distributive unwinding” of double helix which depends on single-stranded DNA. Therefore, recA protein promotes unwinding of the double helix by either of two distinct pathways. Stress caused by the processive unwinding could explain the dissociation of D-loops and reversible inactivation of the double-stranded DNA in a D-loop cycle.

recA protein is a polypeptide of 40,000 daltons and is coded by the recA gene of Escherichia coli (1-3). This protein is indispensable for general genetic recombination in this orga-

* This work was supported in part by a grant for “Life Sciences” to Riken Institute from the Science and Technology Agency of Japan and by a grant from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address, Tokyo Research Institute, Seikagaku Kogyo, Co., Ltd., Higashi Yamatoshi, Tokyo 189, Japan.

3 To whom correspondence should be addressed. TI Present address, Hamari Chemicals Ltd., Osaka-shi, 533, Japan. 11 Present address, College of Agriculture and Veterinary Medicine,

Nihon University, Setagaya-ku, Tokyo 154, Japan.

nism (4) and plays a direct role (5). recA protein has DNA- dependent ATPase activity (6, 7) and exhibits a wide variety of ATP-dependent activities in uitro: (i) single-stranded DNA-dependent endopeptidase (8, 9), (ii) pairing of homologous DNA molecules, if one of them has a single-stranded region (10-14), (iii) strand exchange (15, 16), (iv) unidirectional elongation of heteroduplex joints (17-19), (v) dissociation of D-loops (20, 21), and (vi) unwinding of the double helix (22, 23). The endopeptidase activity is related to a regulatory role of recA protein in “SOS functions” (see Refs. 24 and 25 for review). Although recA protein is isolated from a prokaryote, the activities to promote homologous pairing, strand exchange, and elongation of heteroduplex joints can explain the central steps of general recombination in eukaryotes (see Ref. 26). This view is supported by the recent finding that a protein with very similar poperties to those of recA protein was isolated from a eukaryote, Ustilago maydis

ATP+,’ an analogue of ATP, is a competitive inhibitor of DNA-dependent ATPase activity of recA protein and is not hydrolyzed significantly by the protein (28, 29). In the presence of ATPyS, recA protein does not promote homologous pairing of DNA molecules, but was shown to unwind the double helix (30, 31). In the standard reaction buffer for D- loop formation, the ATPyS-dependent unwinding also depends on the presence of single-stranded DNA (30, 32, 33). Since heterologous as well as homologous single-stranded DNA is effective as a cofactor for the ATPyS-dependent unwinding, the mechanism of aligning the two DNA molecules at homologous sites in homologous pairing by recA protein can be explained by this single-stranded DNA-dependent unwinding (see Refs. 28 and 30).

On the other hand, the negative superhelicity of DNA is supposed to play a role in genetic recombination (see Ref. 34 for review). When recA protein is incubated with negative superhelical double-stranded DNA formed in uiuo (form I DNA)’ in the presence of homologous single-stranded DNA fragments and ATP, some of the reactions promoted by recA protein make a cycle that we call a D-loop cycle (Fig. 1; see Ref. 35). The D-loop cycle consists of (i) pairing of homologous DNA molecules to form D-loops, (ii) dissociation of the D-loops and inactivation of the form I DNA as substrate for

The abbreviation used is: ATP-yS, adenosine 5’-0-(3-thiotriphos- phate).

*The forms of double-stranded DNA are designated as follows: form I DNA, closed circular DNA with a natural negative linking difference (which is formed in uiuo); form I1 DNA, circular double- stranded DNA with one or more single-stranded breaks; form IV DNA, fully relaxed closed circular DNA; form X DNA, closed circular DNA with a much larger negative linking difference than that of form I DNA.

(27).

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ATP-dependent Unwinding of Double Helix by E. coli recA Protein 12395

D-loop formation, and (iii) reactivation of the inactivated form I DNA. The dissociation of D-loops was shown to be a polar process (36). The extensive unwinding is observed after the formation and dissociation of D-loops in form I DNA and is responsible for the inactivated state of form I DNA as a substrate in D-loop formation (22). T h e dissociation of D- loops in form I DNA by recA protein could be explained by the following model (21, 35; Fig. 1). The unwinding of the double helix starts at t h e site of a D-loop and proceeds unidirectionally (5' to 3' of the D-loop). This unidirectional unwinding causes positive supercoiling of form I DNA, which stimulates rewinding at the trailing end of the unwound region (i.e. D-loops). In this way, the unwound region in form I DNA migrates unidirectionally along the molecule. When the rewinding portion migrates beyond the 3' terminus of single- stranded DNA in the D-loop, the D-loop dissociates.

The unwinding that is assumed in this model has the following features. It is (i) initiated at the site of a D-loop and (ii) propagated by a processive reaction. In this study, we show that extensive ATP-dependent unwinding of the double helix is likely to be initiated at the si te of a D-loop and that the unwinding is propagated by a processive reaction.

MATERIALS AND METHODS

Enzymes-recA protein used in this study was fraction V (DEAE- cellulose fraction) prepared as described (32,37). The amount of recA protein is expressed in moles of polypeptide of 40,000 daltons. Yeast topoisomerase I, which was a gift from T. Iino and Dr. H. Watabe (this department), was purified from a cell-free extract of Saccharo- myces cereuisiae IAM4274 (see Ref. 22). Pyruvate kinase was pur- chased from Worthington.

DNA-Circular double-stranded 13H]DNA and circular single- stranded DNA from phage fd and dX174am3 were purified as described previously (38). Fragments of single-stranded DNA were prepared by boiling as described elsewhere (30,32). The average size of single-stranded fragments of fd and 4x174 was 200 nucleotides. Form IV DNA was prepared by relaxing form I DNA with yeast topoisomerase I at 37 "C in the standard reaction buffer for the first incubation without single-stranded DNA, recA protein, and ATP. Concentrations of DNA are expressed in moles of nucleotide residues/ liter and denoted by M. In some experiments (shown in Figs. 4 and 7 and in Tables I and II), the concentrations of DNA are expressed in moles of DNA molecules/liter and denoted by M ~ . M~ is based on 6,806 and 12,800 nucleotide residues/one DNA molecule for circular single-stranded phage DNA and circular double-stranded DNA, respectively, of fd (39), 10,800 nucleotide residues/one DNA molecule for 6x174 circular double-stranded DNA (40), and 200 nucleotide residues/one DNA molecule for single-stranded fragments.

Assay of Unwinding of the Double Helix-In principle, we compared the average linking number of closed circular double-stranded DNA relaxed by a eukaryotic topoisomerase I in the presence of double helix unwinding reagents with that of closed circular double-stranded DNA relaxed in their absence. The difference between the linking numbers of the former and the latter (the linking difference3) directly indicates the angle of unwinding by the reagent (41, 42). The formation of an extensively underwound form (form X) of closed circular double-stranded DNA by the combined actions of recA protein and topoisomerase indicates that recA protein extensively unwinds the double helix. The increase in the amount of form X indicates the initiation of extensive unwinding of the double helix, and increase in

To express the topological states of closed circular double- stranded DNA, we use the notation of Liu and Wang (59). Linking difference ( A a ) is defined as Aa = a - a0, where a is the linking number of the DNA and a. is the linking number of the same DNA that is relaxed under standard conditions (0.2 M NaC1, 37 "C, and neutral pH; see Ref. 50). The superhelical density is the linking difference/lO base pairs. DNA with a negative linking difference is termed underwound, and DNA with a positive linking difference is termed overwound (see Refs. 50 and 69 for review). The underwound DNA is negatively superhelical in the absence of DNA unwinding reagents.

the negative linking difference indicates propagation of the unwinding (Fig. 1).

The standard reaction mixture for the first incubation (the higher M$+ condition) contained, in 18 pl, 31 rnM Tris-HC1 (pH 7.5), 15 mM MgC12, 1.5 mM ATP, 2.1 mM dithiothreitol, 100 pg of bovine serum albumin/ml, 11 p~ form I [3H]DNA of fd, 0.11 pM single- stranded fragments of fd (homologous) or 4X174am3 (heterologous), 1.7 p~ recA protein, and an ATP-regenerating system consisting of 3.4 mM phosphoenolpyruvate and pyruvate kinase (28 units/ml), unless otherwise stated. The reaction mixture was incubated at 37 "C (the first incubation). Under the lower M e condition, the concentration of MgClz was decreased to 1.2 mM.

Then, 2 p1 of yeast topoisomerase I in buffer D (50 mM Tris-HC1 (pH 7.5), 0.3 mM EDTA, 5 mM dithiothreitol, and 10% glycerol) were added, and the mixture was incubated (the second incubation) for 30 s (unless otherwise stated) at 37 "C. When the first incubation was carried out under the lower MgZ+ condition, 1 pl of 0.24 M MgClz was added before the addition of yeast topoisomerase I. The amount of topoisomerase used in the second incubation was that which relaxed all the 11 p~ fd form I DNA in 20 s at 37 "C in the standard reaction buffer without ATP. After the second incubation, all the reaction mixture was put onto a cold mixture of 5 p1 of 0.1 M EDTA (pH 9), 5 p1 of phenol saturated with 10 mM Tris-HC1 (pH 8.0), and 1.5 pl of 10% Sarkosyl (Ciba-Geigy, NL97) and chilled in an ice-water bath. The sample was analyzed by agarose gel electrophoresis or isopycnic centrifugation in CsC1-ethidium bromide.

Agarose Gel Etectrophoresis-Samples were subjected to electrophoresis on 1% agarose gel in buffer E (43) in the absence of dye at 1.7 V/cm for 15 h at room temperature. After electrophoresis, the gel was stained with 0.5 pg/ml of ethidium bromide for 30 min at room temperature and photographed as described (32). Then, each lane of gel was cut into several pieces, and each was dissolved by boiling for 4 min with 0.1 N HC1, applied to a glass filter (Whatman GF/C), and dried. Radioactivity on the filter was assayed in a Beckman LS8100 liquid scintillation counter in toluene scintillator. Values are given as percentage of the total radioactivity recovered from each lane. Form X DNA (Fig. 3A, h n e s 3 to 6) migrated as a band faster than form I DNA (Fig. 3A, a major band in lane I ) . Form IV DNA (Fig. 3A, lane 2) migrated much slower than form I DNA and gave a ladder of bands near the band of form I1 DNA (Fig. 3A, top minor band in lunes 1 to 6) .

Isopycnic Centrifugation in CsC1-Ethidium Bromide-The sample was diluted with 10 mM Tris-HC1 (pH 8.0) containing 1mM EDTA, 500 pg of ethidium brornide/ml and CsCl to a density of 1.575 g/cm3. Centrifugation was carried out in a Hitachi RP55 rotor at 37,000 rpm for 70 h at 20 "C. Fractions were collected from the bottom of the tube and applied to glass filters (Whatman GF/C). The filters were treated with 5% trichloroacetic acid for 15 min at room temperature and washed twice with 5% trichloroacetic acid and then twice with ethanol. Radioactivity on the filter was counted as described above.

D-loop and Nicking Assays-D-loop assay and assay of the amount of nicked double-stranded DNA were as described or cited previously (32). Treatment with Sarkosyl was carried out in an ice-water bath for 30 min.

RESULTS

Formation of Extensively Underwound DNA3 by the Com- bined Action of recA Protein and Yeast Topoisomerase I- Form I DNA of phage fd, homologous single-stranded fragments, recA protein, and ATP were incubated at 37 "C for 45 min (the first incubation) in the standard reaction mixture for D-loop formation (the higher MgZ+ condition, containing 15 mM MgC1,) to complete the sequential formation and dissociation of D-loops (Fig. 2 A , open circles). Then, excess yeast topoisomerase I was added to the reaction mixture, and incubation was continued at 37 "C (the second incubation) for 20 to 60 s. Yeast topoisomerase I can relax both positive and negative supercoiling (see Ref. 22). The amount of topoisomerase I used in the second incubation was enough to relax all the form I DNA in the reaction mixture within 20 s at 37 "C. The products were analyzed by agarose gel electrophoresis after treatment with detergent and phenol. During the second incubation with topoisomerase I, form I DNA was converted to a form that migrated faster than untreated form


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12396 ATP-dependent Unwinding of DG

f a

1 recA + Top0

1 recA + Top0

FIG. 1. Schematic diagram of experiments to demonstrate the initiation and propagation of ATP-dependent unwinding of the double helix by recA protein. The unwinding by recA protein is initiated at the site of a D-loop (in ii.) or at a transiently denatured site (in i,) in form I DNA and propagated unidirectionally. The unidirectional propagation (indicated by an arrow with a solid tail; see Ref. 35 and “Discussion”) causes positive supercoiling of the form I DNA (iiiJ, which promotes the rewinding at the trailing end of the unwound region (indicated by an arrow with a broken hail). The size of the unwound region (or the extent of the unwinding) is limited by the positive supercoiling. By this process, the unwound region migrates along the strand of the form I DNA. When the unwinding is initiated at the site of a D-loop, the D-loop is dissociated by the unidirectional migration of the unwound region (step from ii, to iii.). D-loop cycle (35) is a cycle of sequential reactions consisting of the formation of D-loops ( i to iiJ, the dissociation of the D-loops, and inactivation of the form I DNA (ii. to iii.) and the reactivation of the form I DNA (iii,, to i, a direct pathway not indicated in this figure). The form I DNA that is not unwound (i) is converted to form IV by yeast topoisomerase I, but the form I DNA that is unwound and positively supercoiled by recA protein (iii,,) is converted to form X (iu,) by the action of the topoisomerase (Topo). The deviation of the linking number of form X (or form I) from that of form IV is the linking difference of form X (or form I). During prolonged incubation with topoisomerase, the unwound region extends by propagation of unwinding without topological limitation (step from iu. to u.). The unwinding of the double helix by 360” in the presence of the topoisomerase results in increase of the negative linking difference (-An) by 1. After all proteins are removed (step d) , form X (iu and u ) is much more negatively superhelical than form I DNA (i). Increase in the amount of form X indicates the kinetics of the initiation of extensive unwinding (step from i, to iii. or step from ii. to io), and increase in the negative linking difference of form X DNA in the presence of the topoisomerase indicates the rate of propagation of the unwinding (step from iu, to uJ. Unwound region is indicated by a region of a pair of concentric circles with a larger distance in this figure. However, unwinding observed in this study does not necessar- ily mean denaturation, but means decrease in rotation angle of double

uble Helix by E. coli recA Protein

I DNA (Fig. 3A). As described in detail in the later section, on very short treatment (within 1 min) with the topoisomerase I, almost all the form I DNA was converted to a form with 2 to 3 times greater negative superhelical density than untreated form I DNA (Fig. 8F, closed circles). We call this form of DNA with a larger negative superhelical density than the natural form I DNA form X for simplicity. Formation of the form X DNA indicates that recA protein extensively unwound the double helix and accumulated positive supercoiling in the form I DNA (see Ref. 22).

This extensive ATP-dependent unwinding consisted of two distinct steps: initiation and propagation. The former step was observed on increase in the amount of form X DNA, as described in the next section. The latter step was observed on the increase in the negative linking difference3 of the form X DNA in the presence of eukaryotic topoisomerase I, and will be dealt with in the latter sections.

Kinetics of Single-stranded DNA-dependent Initiation of Extensive Unwinding of the Double Helix by recA Protein- The amount of form X indicates the amount of form I DNA that is unwound by recA protein. The amount of form X was not significantly changed in at least 8 min during the second incubation (treatment with topoisomerase) (Fig. 3B). There- fore, the increase in amount of form X shown in Fig. 2A indicates the kinetics of initiation of extensive ATP-dependent unwinding during the first incubation of form I DNA with homologous single-stranded fragments, recA protein, and ATP. As shown in Fig. 2 4 , the amount of D-loops increased and then decreased (open circles), but the fraction of form I DNA in the unwound state (closed circles) increased gradually until the dissociation of D-loops was complete. Finally, almost all the form I DNA was unwound. These data are consistent with an idea that the D-loop precedes the unwound state of form I DNA.

Effect of Single-stranded DNA on Initiation of Extensive Unwinding of the Double Helix-The initiation of unwinding was greatly stimulated by homologous single-stranded fragments (Figs. 2 and 4, A and B, closed circles). When fd form I DNA was replaced by 4x174 form I DNA, single-stranded fragments of 4x174 were effective as a cofactor in initiation of extensive unwinding, but fd single-stranded fragments were not (Table I). This confirms that, as in the case of D-loop formation, the initiation of unwinding was truly stimulated by homologous single-stranded fragments. Form I DNA and homologous circular single-stranded DNA are paired by recA protein to form “nascent D-loops” in which the single- stranded DNA and its complementary strand of the form I DNA pair without net topological intenvinding, but cannot form “mature D-loops” (12,14,20,36), since recA protein has neither topoisomerase activity nor endonuclease activity (10, 30, 32). Homologous circular single-stranded DNA also supported the initiation of extensive unwinding with the same efficiency as fragments of homologous single-stranded DNA (Fig. 4A). These results indicate that the initiation of unwinding involves the recognition of sequence homology between double-stranded DNA and single-stranded DNA. The recognition of sequence homology is likely to be carried out by formation of the hydrogen bonds of complementary bases between two DNA molecules, i.e. by the formation of either nascent D-loops or mature ones.

Heterologous single-stranded DNA, either circular or frag- mented, stimulated the initiation of unwinding, although its

helix. Electron microscopic studies in the presence of ATP-@ (31) indicated that, in unwound region by recA protein, both strands of double-stranded DNA did not extensively separated.


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ATP-dependent Unwinding of Double Helix by E. coli r e d Protein 12397

1st incubation, min 1st incubation, min FIG. 2. Time course of initiation of extensive unwinding of the double helix by recA protein. The

increase in the amount of form X indicates the kinetics of the initiation of extensive unwinding. A, with form I DNA. fd form I [3H]DNA (82% form 1 and 18% form 11) and single-stranded fragments (0.11 p ~ , 0.57 X 10- MC,) were incubated with recA protein (1.7 p ~ ) in the standard reaction mixture for the indicated times at 37 "C (the first incubation). Subsequently, yeast topoisomerase I was added, and the mixture was incubated for 30 s at 37 "C (the second incubation). Samples were analyzed for form X by agarose gel electrophoresis (closed symbols). Samples without the second incubation were assayed for D-loops (open symbols). 0 and 0, fd single-stranded fragments; + and 0, 6x174 single-stranded fragments; A and A, without single-stranded DNA; V, without recA protein; W, without ATP. E, with form IV DNA. fd form IV [3H]DNA (60% form IV, 1% form I, and 39% form 11) and single- stranded fragments (1.1 p ~ , 5.7 X lo-' MO) were incubated with 4.6 pM recA protein and 3.5 mM ATP for the indicated times at 37 'C (the first incubation). An ATP-regenerating system was omitted. Then, yeast topoisomerase I was added, and the mixture was incubated for 5 min at 37 "C (the second incubation). The amount of form X was assayed by agarose gel electrophoresis. It might be expected that formation of D-loops in form IV DNA would be inhibited by positive supercoiling. However, addition of topoisomerase (the same amount as that in the second incubation) to the first incubation did not significantly affect the rate of initiation of extensive unwinding. 0, fd single-stranded fragments; +, 4x174 single-stranded fragments, A, without single-stranded DNA; 0, without ATP V, without recA protein.

A

1 2 3 4 5 6

- 0 10 20 3( 2nd incubation, min

FIG. 3. Formation of the extensively underwound form (form X) by the second incubation with yeast topoisomerase I. A, fd form I ['HIDNA (88% form I DNA and 12% form I1 DNA) on fd single-stranded fragments were incubated with recA protein in the standard reaction mixture (the higher M k condition) for 40 min (the first incubation). Then, yeast topoisomerase was added, and the mixture was incubated for the indicated times at 37 "C (the second incubation). Samples were analyzed by agarose gel electrophoresis. Lane 1, untreated form I DNA; lane 2, without the first incubation and 5 min of the second incubation; lanes 3 to 6, with the first incubation and the second incubation for 20 s (lane 3), 60 s (lane 4), 120 s (lane 5), or 100 min (lane 6). B, fractions of form X DNA and form I1 DNA are plotted against the time of the second incubation. Open symbols. the first incubation and the second incubation were carried out for 40 min at 37 'C as in A. Closed symbols; fd form I 13H]DNA (72% form I and 28% form 11) and recA protein were incubated in the absence of single-stranded DNA under the lower Mg2+ condition for 20 min at 37 "C (the first incubation). Then, MgC12 (to give the higher M e condition) and yeast topoisomerase I were added, and the mixture was incubated for the times indicated at 37 "C (the second incubation). Samples were analyzed by gel electrophoresis. 0 and 0, form X A and A, form 11.


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12398 ATP-dependent Unwinding of Double Helix by E. ATP-dependent Unwinding of Double Helix by E.

9 0

.- r 0 2 - al I)

L.-, > 0.4 (x8 0 4 8

coli recA Protein

0 2 4 6 Single -stranded DNA

MO X 109 FIG. 4. Effect of the concentration of single-stranded DNA on the initiation of extensive unwinding

of the double helix by recA protein. The rate of increase in the amount of form X indicates the rate of initiation of extensive unwinding. A and B, with form I DNA. fd form I [3H]DNA (11 p ~ , 8.7 X 10"' M ~ ; 78% form I and 22% form 11) and the indicated amounts of single-stranded DNA were incubated with recA protein for 15 min (with homologous single-stranded DNA) or 40 min (with heterologous single-stranded DNA) at 37 "C (the first incubation). Then, yeast topoisomerase I was added, and the mixture was incubated for 30 s (with single- stranded fragments) or 8 min (with circular single-stranded DNA). Samples were assayed for form X by agarose gel electrophoresis. Under the conditions used in these experiments, the amount of form X was proportional to the time of the first incubation (see Fig. 2 A ) . a, fd single-stranded fragments; +, 4x174 single-stranded fragments; 0, fd phage circular single-stranded DNA; 0, 6x174 phage circular single-stranded DNA. C, with form IV DNA. fd form IV [3H]DNA (11 p ~ , 8.7 X 10"' M'; 60% form IV, 1% form I, and 39% form 11) and the indicated amounts of single-stranded fragments were incubated with 4.6 FM recA protein and 3.5 mM ATP for 20 min at 37 "C (the first incubation). An ATP-regenerating system was omitted. Then, yeast topoisomerase I was added, and the mixture was incubated for 5 min at 37 "C (the second incubation). The amount of form X was assayed by agarose gel electrophoresis. The broken line indicates the initial velocity of initiation of extensive unwinding of form I DNA by recA protein (1.7 p ~ ) , for comparison. The data with form I DNA were taken from A.

TABLE I Effect of homology between form I DNA and single-stranded

fragments on the initiation of extensive unwinding of the double helix by recA protein

Form I DNA Single-stranded frag- Initial velocity of ini-

ments tiation of extensive unwinding'

fd M ~ / x 10'3

fd 3.6 4x174 0.17 None 0.22

4x174 4x174 2.4 fd 0.1 None 0.1

' The increase in the amount of form X indicates the kinetics of initiation of extensive unwinding of the double helix. Form I 13H] DNA of fd (11 p ~ , 8.7 X 10"' M ~ ; 80% form I DNA and 20% form I1 DNA) or 4x174 (11 p ~ , 10 X 10"' M ~ ; 55% form I DNA and 45% form I1 DNA) and single-stranded fragments (0.11 p ~ , 0.57 X lo-' M ~ ) were incubated with recA protein for 7.5 or 15 min (with homologous single-stranded fragments) or for 40 or 55 min (with heterologous single-stranded fragments or without single-stranded DNA) at 37 "C (the first incubation). Then, yeast topoisomerase I was added and the mixture was incubated for 5 min. Under the conditions used in these experiments, the amount of form X was proportional to the time of the first incubation (see Fig. 2 A ) . The amount of form X was assayed by agarose gel electrophoresis and did not significantly change in 8 min during the second incubation (Fig. 38).

capacity to support the initiation was '120 to %oo of that of homologous DNA (Fig. 4, A and B, open and closed diamonds, respectively).

Effect of Superhelicity on the Initiation of Extensive ATP- dependent Unwinding-Negative superhelicity is not required for the initiation of the extensive unwinding in the presence of homologous single-stranded DNA, but greatly accelerates the initiation. As shown in Figs. 2B and 4C (closed circles), form X was derived from relaxed closed circular double-

stranded DNA (form IV DNA) in the presence of homologous single-stranded fragments but not in their absence or in the presence of heterologous fragments. The initial rate of initiation in form IV DNA was less than %S of that in form I DNA (Fig. 4C), as in the case of D-loop formation, i.e. negative superhelicity of form I DNA accelerates the formation of D-

Initiation of Extensive ATP-dependent Unwinding of the Double Helix in the Absence of Single-stranded DNA-The polar dissociation of D-loops suggests that recA protein prop- agates unwinding of the double helix by a processive reaction, as described in the Introduction. This suggestion can be confirmed by the demonstration that unwinding is propagated under conditions that do not allow its initiation, e.g. in the absence of single-stranded DNA.

Our previous results suggested that unwinding of the double helix by recA protein was associated with extensive hydrolysis of ATP (22, 44). On the other hand, at neutral pH, form I DNA supports the hydrolysis of ATP by recA protein two thirds as well as single-stranded DNA in the presence of 1 to 2 mM MgClz (the lower M$+ condition), but little in the presence of more than 4 mM MgClz (10,32). Therefore, ATP- dependent unwinding should occur in the absence of single- stranded DNA, when the concentration of MgC12 is decreased from about 13 mM (the standard concentrations for D-loop formation, the higher M F condition) to 1 to 2 mM. This was found to be the case. recA protein, form I DNA, and ATP were incubated in the absence of single-stranded DNA under the lower Mg2+ condition at 37 "C (the first incubation). After various times, the reaction mixture was adjusted to the higher M$+ condition, excess yeast topoisomerase I was promptly added, and incubation was continued a t 37 "C (the second incubation) for 30 s. As shown in Fig. 5A, under the lower Mg2+ condition, extensive unwinding was initiated very quickly in the absence of single-stranded DNA (closed triangles) in the first incubation, and within 15 min unwinding

loops 30 to 50-fold (21).


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ATP-dependent Unwinding of Double Helix by E. coli recA Protein 12399

80 I A

0 20 40 60 1st incubation, min

80 l B " I m- I I " C 40

I- I

!",*s. , I O O 20 40 60

1st incubation, min

FIG. 5. Time course of single-stranded DNA-independent initiation of extensive unwinding of the double helix by recA protein in the lower Mg2+ condition. A, increase in the amount of form X indicates the kinetics of initiation of unwinding. fd form 1 [3H]DNA (80% form I and 20% form 11) was incubated with recA protein in the absence of single-stranded DNA under the lower MgZf condition for the indicated times at 37 'C (the first incubation). Subsequently, the reaction mixture was adjusted to the higher MgZf condition. Then, yeast topoisomerase I was promptly added, and the mixture was incubated for 30 s at 37 "C. The amount of form X was assayed by agarose gel electrophoresis. A, complete system; V, without recA protein; ., without ATP; A, first incubation under the higher M$+ condition, B, fd form I [3H]DNA and 1.1 pM single-stranded fragments were incubated with 3.6 PM recA protein under the lower Mg2f condition for the indicated times at 37 "C. The amount of D- loops was determined by D-loop assay.4 A, without single-stranded DNA; 0, fd single-stranded fragments; +, 6x174 single-stranded fragments; V, without recA protein.

was initiated in 85% (68/80) of the input form I DNA. The initiation of unwinding required ATP as well as recA

protein (Figs. 5A and 6). The formation of D-loops is not linearly dependent on the concentration of recA protein, and no D-loops are formed unless a certain level of recA protein was present (10, 11, 20, 21, 28). The amount of recA protein required for D-loop formation is proportional to the concentration of single-stranded DNA (20, 21). Then, we increased the amount of form I DNA about 8-fold to make clear a threshold of the amount of recA protein required for the reaction, if any. Unlike in the case of D-loop formation, in the absence of single-stranded DNA, the initial velocity of initiation of extensive unwinding was linearly dependent on the concentration of recA protein, and no threshold was detected (Fig. 6).

'The amount of D-loop is the difference between the value obtained with homologous single-stranded DNA and that with heterologous single-stranded DNA. The small increase in the value obtained by the D-loop assay in Fig. 5B is due to DNase activity contaminating the preparat,ion of pyruvate kinase.

S I * I S 40

x E L $ 2 0 -

/*/ 4- 01

0 2 4 6 8

40

x E L $ 2 0 -

/*/ 4- 01

0 2 4 6 8 RecA protein, yM

FIG. 6. Linear dependence of the initiation of extensive unwinding on the concentration of recA protein. fd form I 13H] DNA (80 PM; 86% form I DNA and 14% form I1 DNA) and the indicated amount of recA protein were incubated in the absence of single-stranded DNA under the lower Mg2f condition for 5 min at 37 "C (the first incubation). Then, MgC12 (to give the higher MgZf condition) and yeast topoisomerase were added, and the mixture was incubated for 30 s at 37 "C. The amount of form X was assayed by agarose gel electrophoresis. The rate of increase in the amount of form X was constant during the first 5 min in the first incubation under the conditions used in this experiment, and, therefore, indicates the initial velocity of initiation of extensive unwinding. 0, complete system; A, without the first incubation; ., without ATP.

Role of Superhelicity in the Initiation of Extensive Unwind- ing in the Absence of Single-stranded DNA-When form I DNA was replaced by relaxed closed circular DNA (form IV DNA), the initial rate of single-stranded DNA-independent initiation of extensive unwinding decreased to %OO of that with form I DNA (Table 11). Form I DNA has transient denatured sites (45-48), and these may play a role in initiation of extensive ATP-dependent unwinding in the absence of single-stranded DNA.

Although, under the higher Mg2+ condition, extensive unwinding of form I DNA by recA protein starts mainly at the site of a D-loop, we have shown that initiation of extensive unwinding occurs very slowly in the absence of single- stranded DNA under this condition (Ref. 22; Fig. 5A, open triangles). The single-stranded DNA-independent initiation of extensive unwinding favors the lower Mg2f condition as described above, supporting the role of transient denatured sites in the initiation, since lower ionic strength favors denaturation of the double helix (49).

Then, we tested two possibilities about the role of the denatured site. (i) Under the lower Mg2+ condition, the denatured region in form I DNA might act as a homologous single-stranded DNA and support formation of a nascent D- loop which could serve as an initiation site of ATP-dependent unwinding. (ii) Alternatively, the denatured site itself, rather than D-loops, might serve as the initiation site of extensive unwinding. If the former possibility is true, the initiation of extensive unwinding should be a second order reaction with respect to form I DNA. On the other hand, if the latter is true, the initiation of extensive unwinding should be a first order reaction. The result in Fig. 7 clearly indicates that the rate order of the initiation with respect to form I DNA was 1 and supports the second possibility. The following observations also supports this view. (i) Under the lower Mg2+ condition, we could not detect any formation of D-loops in form I DNA (Fig. 5B; also see Refs. 20 and 32). (ii) Homologous single-stranded DNA could support the initiation of extensive


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12400 ATP-dependent Unwinding of Double Helix by E. coli recA Protein

TABLE I1 Effect of superhelicity on the extensive unwinding of the double helix

bv recA Drotein Initial veloc-

~~~

Double-stranded Single-stranded frag- ity of initia- MgCI, tion of exten-

sive un- DNA ments

windinp M ~ I S x 1013

Form I None LoweP 20

Form IV None Lower 0.10 Homologous 0.10 Heterologous 0.10

Form IV None Higher 0.036 Homologous 1.4 Heterologous 0.06

"The increase in the amount of form X indicates the rate of initiation of extensive unwinding of the double helix (Fig. 1). fd form I [3H]DNA (11 PM, 8.7 X 10"' M ~ ; 55% form1 and 45% form 11) or fd form IV [3H]DNA (11 pM, 8.7 X 10"' MO; 60% form IV and 40% form 11) and 4.6 PM recA protein were incubated with or without 1.1 PM (5.7 X lo-' MO) single-stranded fragments of fd (homologous) or @X174 (heterologous) for 2 min (with form I DNA) or 40 min (with form IV DNA) at 37 "C (the first incubation). When the first incubation was carried out under the lower M%+ condition, MgClz was then added to the reaction mixture to give the higher M%+ condition. Then, yeast topoisomerase I was promptly added, and the mixture was incubated for 5 min at 37 "C (the second incubation). Under the conditions used in these experiments, the amount of form X was proportional to the time of the first incubation and did not change significantly in 30 min during the second incubation (Fig. 3B).

'The concentration of MgC12 in the first incubation: lower, 1.2 mM; higher, 15 mM.

unwinding of form IV DNA under the higher M e condition (Table 11; see also Figs. 2 and 4) but could not support initiation of extensive unwinding of form IV DNA under the lower Mg2+ condition (Table 11). These results indicate that, under the lower Mg2+ condition, single-stranded DNA could not support initiation of extensive unwinding by formation of

Propagation of Unwinding of the Double Helix by recA Protein in the Presence of Topoisomerase I-Then, we exam- ined the propagation of unwinding in the absence of its initiation by an assay using yeast topoisomerase I (see Fig. 1). fd form I DNA, recA protein, and ATP were incubated in the absence of single-stranded DNA under the lower Mg2+ condition at 37 "C for a period that gave the maximal yield of form X (the first incubation). After the first incubation, the concentration of MgClz was adjusted to 13 mM (the higher MgZ+ condition) to prevent the initiation of unwinding, and then excess yeast topoisomerase I was promptly added and incubation was continued (the second incubation) at 37 "C for various times. After the second incubation, DNA samples were treated with detergent and phenol to remove proteins and analyzed by agarose gel electrophoresis and the ethidium bromide-buoyant density method (50). As shown in Fig. 3B (closed circles), 71% (52173) of the closed circular DNA was converted to a form (form X) that migrated faster than form I DNA on agarose gel electrophoresis. The rate of migration of form X DNA increased with an increase in the period of the second incubation (data not shown), as expected if the negative linking difference increases during incubation with the topoisomerase. This analysis also revealed that both the fraction of form X and the fraction of nicked circular double- stranded DNA (form I1 DNA) in the sample were constant regardless of the period of the second incubation (Fig. 3B, closed circles). The latter fact indicates that net initiation of

D-loops.

a 3 t

O O L Form 4 I DNA, Mo x l 0 ' O 8

FIG. 7. Linear dependence of the initial rate of initiation of extensive unwinding of the double helix on the concentration of double-stranded DNA. The indicated amounts of fd form I [3H] DNA (71% form I and 29% form 11) and 3.3 PM recA protein were incubated in the absence of single-stranded DNA under the lower M%+ condition for 5 min at 37 "C (the first incubation). Then, MgCI2 (to give the higher M%+ condition) and yeast topoisomerase I were added, and the mixture was incubated for 30 s at 37 "C. The amount of form X was assayed by agarose gel electrophoresis. Since the amount of form X was proportional to the time of the first incubation during the first 5 min, the increase in the amount of form X indicates the initial rate of initiation of extensive unwinding. The ratio ( k in s-') of the initial velocity (in MO/S) to the concentration of form I DNA (in M ~ ) is plotted against the concentration of form I DNA. If the rate order of the initiation with respect to form I DNA is 1, the ratio ( k ) is constant regardless of the concentration of form I DNA. If the rate order is 2, k is proportional to the concentration of form I DNA.

extensive unwinding did not occur during the second incubation and that closed circular DNA was not significantly nicked during the second incubation.

As shown in Fig. 8, B to D , form X DNA has a homogeneous superhelical density. After 15 min of the second incubation, most of the double-stranded DNA, which consisted of form X and the smaller amount of form 11, formed a peak at or near the density of form I1 DNA and a minor part formed a peak a t a density of form IV DNA in a CsCl density gradient containing ethidium bromide (Fig. 8 D ) . The distance from the center of the rotor to the median of the peak of closed circular double-stranded DNA decreases as the linking number decreases. This distance is plotted against the period of the second incubation in Fig. 8E. We calculated the superhelical density of form X DNA from the data shown in Fig. 8E and data in other experiments using the equation of Burke and Bauer (see Ref. 50) and assuming that this equation is valid in the range of superhelicity of form X (Fig. SF). During the initial 1 min of the second incubation, the negative linking difference of form X increased quickly. The calculated superhelical density of fd form X DNA at 1 min of the second incubation was 3.5 times that of fd form I DNA. Since topoisomerase I in the amount used in these experiments took 20 s to remove all superhelical turns of form I DNA at 37 "C, the first quick increase in the negative linking difference (Fig.

seems not to indicate the rate of unwinding by recA protein, but rather the kinetics of relaxing positive supercoiling in the unwound form I DNA. Later than 1 min of the second incubation, the rate of increase in the negative linking difference slowed down but the increase continued for a t least 15 min. After 15 min in the second incubation with topoisomerase I in the absence of single-stranded DNA, the form I DNA acquired a sufficient negative linking difference to sat-

T. Ohtani, M. Iwabuchi, and T. Shibata, unpublished observation.


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ATP-dependent Unwinding of Double Helix by E, coli recA Protein 12401

Bottom Fraction of gradient

" 10 20

2nd inchtion, min

01-0 0 4 8

2nd incubation, min

FIG. 8. Time course of propagation of unwinding of double helix by recA protein (analyzed by isopycnic centrifugation in CsC1-ethidium bromide). The first incubation and the second incubation were carried out as described in the legend to Fig. 3B. After Centrifugation, fractions were collected from the bottom of the tube. Total 3H counts recovered from the tubes were 3000 to 4000 cpm. A: *, untreated form I1 DNA; 0, untreated from I DNA; 0, without the first incubation and 1 min of the second incubation. B to D, the first incubation was carried out in the absence of single-stranded DNA under the lower M F condition for 20 min at 37 "C. The second incubation was carried out for 1 min ( B ) , 4 min ( C ) , or 15 min (D) under the higher M e condition at 37 "C. E, the distance from the center of the rotor to the median of the peak of double-stranded DNA was plotted against the time of the second incubation. The second incubation was carried out under the higher Mg2+ condition in either the absence or presence of single-stranded DNA. A, form X, the first incubation was carried out in the absence of single-stranded DNA, under the lower Mg2f condition; 0, form X, the first incubation and the second incubation were carried out in the presence of 0.11 PM fd single-stranded fragments under the higher Mg2+ condition; M, untreated form I DNA; 0, untreated form I1 DNA; V, without the first incubation and 1 min of the second incubation. F, superhelical density was calculated from the data shown in E using the equation of Burke and Bauer (see Ref. 50). The superhelical density of fd form I DNA was taken as -0.065, based on the size of D-loops in fd form I DNA formed in the absence of topoisomerase (Ref. 32; see Refs. 52 and 48). The average negative linking difference was calculated, taking 6408 as the number of base pairs in fd form I DNA (39). Symbols are the same as in E.

urate the DNA with ethidium bromide (Fig. 8E). Therefore, the ATP-dependent unwinding by recA protein was propagated for at least 15 min in the second incubation. Since the conditions used in the second incubation (i.e. the higher Mg2+ condition and the absence of both negative superhelicity and single-stranded DNA) did not allow the initiation of unwinding (Table I1 and Fig. 3B), the propagation of ATP-dependent unwinding was a processive reaction. The presence of homologous single-stranded DNA did not significantly stimulate the propagation of unwinding by recA protein (Fig. 8, E and F) and also did not affect the profile of isopycnic centrifugation in CsC1-ethidium bromide (data not shown).

The ATP-dependent unwinding by recA protein is propagated so long as enough ATP is supplied (35, 44). However,

we could not calculate the maximal negative linking difference of form X DNA obtained by the combined action of recA protein and the topoisomerase I, since we could not measure negative superhelical densities of more than that sufficient to saturate the tested DNA with ethidium bromide.

DISCUSSION

Initiation and Propagation of Extensive Unwinding of Dou- ble Helix by recA Protein-In this study and a previous one (22), we showed that recA protein extensively unwinds the double helix by an ATP-dependent reaction. This extensive unwinding by recA protein consists of two steps: initiation and propagation (Fig. 1). The initiation occurs (i) at D-loop sites in negative superhelical or relaxed double-stranded DNA


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12402 ATP-dependent Unwinding of Double Helix by E. coli recA Protein

or (ii) at transient denatured sites in negative superhelical DNA. Purified D-loops made by uncatalyzed reaction were dissociated by recA protein (20). As discussed below, the dissociation of D-loops is caused by the extensive unwinding; this result confirms that the D-loop is the site of the initiation of unwinding. At the D-loop sites, the two strands of double- stranded DNA are unpaired. Therefore, unpairing of the double helix seems to initiate extensive unwinding by recA protein.

The nascent D-loops serve as the initiation site of the extensive unwinding of form I DNA as well as the mature D- loops (Fig. 4). Wu et al. (23) found that the nascent D-loops (which they called paranemic joints) also initiate the extensive ATP-dependent unwinding of nicked circular double-stranded DNA (form I1 DNA) by recA protein. We suggested a possible role of the D-loop cycle in the recognition and pairing of homologous chromosomes at meiosis in eukaryotes (35). The formation of the nascent D-loops and the initiation of unwinding at nascent D-loop sites indicate that the D-loop cycle can play the suggested role in the absence of breakage of DNA strands as well as in its presence.

We have shown that, through the formation and dissociation of D-loops, almost all the form I DNA is inactivated as a substrate of D-loop formation (21). This study and a previous one (22) show that through the formation and dissociation of D-loops almost all the form I DNA is unwound by recA protein and acquires positive supercoiling. Therefore, the inactivated state is an unwound state. This conclusion is also supported by the observed correlation between full reactivation of the inactivated form I DNA and complete loss of the unwound state by addition of ADP to the reaction mixture (22, 35). The stress caused by unwinding of the double helix readily explains the inactivation of form I DNA, since formation of D-loops requires further unwinding of the double helix and increases the stress (48,51, 52).

Once initiated, the unwinding by recA protein is propagated even under conditions that do not allow its initiation, e.g. in the absence of both single-stranded DNA and negative superhelicity and even under the higher Mg2+ condition. This indicates that the propagation of unwinding is a processive reaction. Therefore, we call the single-stranded DNA-independent propagation of unwinding “processive unwinding.” The processive unwinding explains not only the inactivated state of form I DNA, but also the dissociation of D-loops in the form I DNA, as described in the Introduction (Fig. 1). recA protein dissociates the D-loops formed with single- stranded fragments that have a heterologous sequence at the 5’ end, but not the D-loops formed with single-stranded fragments that have a heterologous sequence at the 3‘ end (36). This supports the idea that the processive unwinding is a polar reaction as proposed in our model and indicates that the direction of the processive unwinding is from 5’ to 3’ in the D-loop in which the unwinding is initiated.

As a mechanism of dissociation of D-loops, one might think that free recA protein loads on form I DNA at the site of a D-loop, unidirectionally moves along the strands, and unwinds it. However, this model is unlikely, since, as described in this paper, unwinding is propagated even after the complete dissociation of D-loops or in the absence of D-loop formation.

The processive unwinding might promote unidirectional elongation or heteroduplex joints formed between linear double-stranded DNA and homologous circular single-stranded DNA; the unwinding of the double-stranded DNA from a site of a heteroduplex joint is followed by winding of the single- stranded DNA and its complementary strand. The observed direction of elongation is also from 5’ to 3’ of the single-

stranded DNA (17, 18), which is the same as the direction of processive unwinding.

Assuming that the increase in the negative linking difference might be constant between 1 and 8 min during the second incubation with topoisomerase, we calculated the rate of the unwinding to be 60”/s from the data shown in Fig. 8F. This number corresponds to full unwinding of about 2 base pairs/s. The rate of unwinding by recA protein was calculated to be at least 1 base pair/s from the rate of dissociation of D- loops (44). The rate of unidirectional elongation of the heteroduplex joints by recA protein was calculated to be 1 to 4 base pairs/s (Ref. 53).6 Two Distinct Pathways of Unwinding of Double Helix by

recA Protein-In the presence of ATP-yS, heterologous as well as homologous single-stranded DNA stimulates the formation of a ternary complex (with respect to large molecules) which consists of recA protein, single-stranded DNA, and double- stranded DNA. The double-stranded DNA in the ternary complex is unwound (30, 32, 33). This unwinding does not require superhelicity but requires either homologous or heterologous single-stranded DNA in standard reaction buffer for D-loop formation (30, 32, 33). Since this single-stranded DNA-dependent unwinding of the double helix occurs at any site along the double-stranded DNA, we call this type of unwinding of the double helix by recA protein “distributive unwinding.” The single-stranded DNA-dependent distributive unwinding can explain the mechanism of aligning two DNA molecules a t homologous sites in homologous pairing by recA protein (28, 30). Kinetic studies on D-loop formation by recA protein indicated that in the presence of ATP, instead of ATP-yS, the ternary complex formed was a rate-limiting intermediate of homologous pairing (54). If the distributive unwinding is a partial reaction of D-loop formation, it is expected that in the presence of ATP this single-stranded DNA-dependent unwinding should be transient, or the life time of each unwinding event should be very short compared with the rate of D-loop formation. This might be why, in the presence of ATP, heterologous DNA-dependent unwinding could be detected, but with very low efficiency, by the current assay method involving a period of incubation with the topoisomerase (Fig. 4, A and B).

The above discussions lead us to the conclusion that recA protein unwinds the double helix through two different pathways: processive unwinding and distributive unwinding. Dis- tributive unwinding includes the following partial reactions (Fig. 9A; see Ref. 28). (i) recA protein in stage 1 is activated by binding of ATP and single-stranded DNA (stage 3 via stage 2). (ii) recA protein in stage 3 can bind to double- stranded DNA to form a ternary complex and unwinds the double-stranded DNA (stage 4). If in the complex, the unwound region of the double-stranded DNA and the single- stranded DNA have homologous sequences to each other, the two DNA molecules pair by hydrogen bonding to form heteroduplex. (iii) Upon hydrolysis of ATP to ADP and inorganic phosphate, the ternary complex dissociates and the recA protein returns to the original stage (stage 1).

The processive unwinding might be a cooperative reaction of recA protein; recA protein cooperatively binds to double- stranded DNA at the leading end of an unwound region and extends the unwound region as described above. This COOP- erative reaction might share some of the partial reactions with the distributive unwinding (Fig. 9B). (i) recA protein in stage 1 is activated to stage 3 by both binding of ATP and direct interaction with recA protein that is in stage 4 and binding at the leading end of an unwound region in the double-

R. Kahn and C. M. Radding, personal communication.


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ATP-dependent Unwinding of Double Helix by E. coli recA Protein

B

12403

A

u b II f 2 I

u 2

interact with the recA protein

in stage 4

dsDNA c;r. dsDNA 4 - i -

FIG. 9. Model for the mechanism of processive unwinding and distributive unwinding of double helix by recA protein. A, distributive unwinding; B, processive unwinding. recA protein in stage 1 is activated to stage 3 by binding of ATP and either by binding of single-stranded DNA (ssDNA) (in A ) or by cooperative interaction with recA protein in stage 4 (in B) , is stimulated to bind to double-stranded DNA (dsDNA), and unwinds the double helix (stage 4). Upon the hydrolysis of ATP, the recA protein returns to stage 1. Numbers in circles indicate stages of recA protein, probably different conformations; dots by numbers indicate binding or direct interaction.

stranded DNA. (ii) The following steps are similar to those in the distributive unwinding, i.e. recA protein in stage 3 binds to double-stranded DNA and unwinds it at a site in front of the leading end of an unwound region. Upon hydrolysis of ATP to ADP and inorganic phosphate, the recA protein returns to stage 1. The direct interaction between molecules of recA protein is supported by the observations that recA protein tends to form a filament even in the absence of DNA (55, 56). Cooperative binding of recA protein with double- stranded DNA in the presence of ATPyS was also demon- strated by electron microscopic studies (55-58).

It has been found that recA protein promotes various DNA reactions in the presence of ATP, such as homologous pairing of DNA molecules, unidirectional elongation of heteroduplex joints, formation of Holliday junctions, dissociation of D- loops, inactivation of form I DNA as a substrate for D-loop formation, and unwinding of the double helix (see Introduc- tion and Ref. 26). This variety of reactions is very surprising, considering the apparently simple structure of recA protein; recA protein consists of a single species of polypeptide of 40,000 daltons (1-3). However, this study suggests that the apparent variety of reactions promoted by recA protein is a result of the combination of substrates and only two types of unwinding activities of recA protein.

Acknowledgments-We thank Tohru Lino and Dr. Hiro-omi Wa- tabe for providing yeast topoisomerase I and Dr. Charles M. Radding and Dr. Anna M. Wu for telling us about their unpublished results.

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M Iwabuchi, T Shibata, T Ohtani, M Natori and T AndoEscherichia coli recA protein in the presence of topoisomerase.

ATP-dependent unwinding of the double helix and extensive supercoiling by

1983, 258:12394-12404.J. Biol. Chem.

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