Uptake of homologous single-stranded fragments by superhelical DNA

J. Mol. Riol. (1977) 116, 8054324

Uptake of Homologous Single-stranded Fragments by Superhelical DNA III. The Product and its Enzymic Conversion to a Recombinant

Molecule

ROGER C. WIEGAND, KENNETH L. BEATTIE WILLIAM K. HOLLOMAN AND CHARLES M. RADDING Departments of Internal Medicine, and Molecular Biophysics and Biochemistry Yale University School of Medicine New Haven, Conn. 06510, U.S.A. (Received 11 January

1977, and in revised form

5 July

1977)

When superhelical DNA (RFI)t of phagas 9X174 or G4 takes up a homologous single-stranded fragment, RF DNA and fragment are linked by as many as 300 base-pairs, and a corresponding length of one strand of the RF1 is displaced, forming

a displacement

loop

(D-loop).

The length

of the base-paired

region

was

fragment that was estimated from the fraction of the associated 32P-labeled resistant to digestion by exonuclease VII, as well as by electron microscopy. Dissociation of the fragment by heating was characterized by a sharp melting curve. The displaced strand of the RF DNA was digested by two endonuoleases that act on single-stranded DNA, the S, nuclease of Aspergillua oryzae and the recBC DNAase of EscAerichia c&i. Acting on complexes, both enzymes converted tile form I [3H]DNA into form II DNA, and left some of the associated 32P-labeled fragment undigested. The remaining 32P-labeled fragment could no longer be displaced by branch migration, as expected if the displaced strand of the RF DNA were digested. The action of S1 nucleate also produced the amount of acid-soluble 3H expected from digestion of the D-loop. Treatment of such digested complexes with polynucleotide ligase covalently linked about 35% of the remaining 32P-labeled fragment to 3H-labeled strands, which proves that S, nuclease digested the D-loop.

1. Introduction In preceding papers we have characterized the reaction of superhelical DNA with homologous single-stranded fragments in vitro and have presented evidence of the possible role of this reaction in genetic recombination (Holloman et al., 1975; Holloman t Terminology G4 DNA

is termed

and abbreviations: RF1

or form

the closed double-stranded I;

circular

duplexes

containing

circular

form of phage $X174 or

one or more

interruptions

are

t,ermed form II. The molecule formed by the uptake of a homologous single-stranded fragment by superhelical DNA is called a complex. The associated single strand is sometimes designated as the third strand or donor strand. As defined by Kasamatsu et al. (1971) the term displacement loop or D-loop is used to describe a particular kind of triple-stranded region in DNA containing 2 paired strands and an unpaired strand, the latter covalently linked at both ends to the rest of the duplex DNA. The symbols I, r, and rC stand, respectively, for the number of nucleotides in a fragment, the number of residues of fragment nucleotide per molecule of circular DNA, and the number of wsidues of fragment nucleotide per molecule of complex. A bar above the symbol indicates a mean value. Concentrations of DNA are given as moles of phosphorus except as indicated. 805

R. C. WIEGAND

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ET AL.

L Radding, 1976; Beattie et al., 1977). In the experiments described here, we have characterized the product, principally by studying the action of several enzymes on complexest made with specific single-stranded restriction fragments. We will discuss the relevance of these enzymological experiments to the possible role of strand uptake in genetic recombination. A preliminary report of some of these observations has been published (Wiegand et al., 1976).

2. Materials and Methods (a) Enzymes and reagents S, nuclease was purified

as described

before by Wiegand

et al. (1975). Pancreatic

DNAase prepared

was purchased from Worthington Biochemicals. Endonuclease R *Hoe111 was from Haemophilus aegyptius by the method of Middleton et al. (1972). The recBC nuclease (fraction VII) of Escherichia coli was the generous gift of Drs D. Eichler and I. R. Lehman (1977). This enzyme is frequently called exonuclease V (Wright et al., 1971) because of its prominent exonuclease activity. However, since the endonucleolytic activity of the same enzyme is crucial to its role in the present experiments, we will call it the recBC DNAase to avoid confusion. Polynucleotide ligase of E. coli was purchased from as described before by Radding (1966). One Biolabs. Phage h exonucleaae was purified unit of activity of S1 nucleate, exonuclease VII, or h exonuclease is the amount of enzyme that renders 10 nmol of DNA nucleotide acid-soluble in 30 min at 37°C under the conditions of the standard assay for each enzyme. One unit of the recBC DNAase is the amount of enzyme which renders 1 nmol of double-stranded DNA acid-soluble in 20 min at 37°C. Acetylated bovine serum albumin prepared by the method of Dowhan (1969) lacked any nuclease activity that cleaved form I DNA of 4X 174 under the reaction conditions for the recBC DNAase. Exonuclease VII was purified 320-fold by a modification of the method of Chase & Richardson (1974a) from strain BW9062 (recBZ1 recC22 sbcBl5 &h-l e&l) which was generously provided by Dr Bernard Weiss of Johns Hopkins University School of Medicine. The cells were lysed with lysozyme by the method of Eichler & Lehman (1977). The extract was dialyzed and chromatographed on DEAE-cellulose and phosphocellulose as described by Chase & Richardson (1974a). There was a single peak of activity on each column. The purified enzyme did not have any endonuclease activity on +X174 RF1 nor did it release detectable acid-soluble material from native h DNA. As originally observed by Chase & Richardson this preparation of enzyme was many times more active on short than on long single-stranded DNA. Carrier-free [32P]phosphoric acid was obtained from New England Nuclear. Thymidine (methyL3H, 6 Ci/mmol) was purchased from Schwarz/ Mann. (b) Preparation

of DNA

RF1 [3H]DNA from phages G4 and 9X174 was prepared by the method of Godson & Boyer (1974). For some preparations the final sucrose gradient in the purification was replaced by gel filtration on a 7 cm2 x 30 cm column of Sepharose 2B in 0.1 M-NaCl, 10 mM-Tris*HCl (pH 7*5), and 1 mM-EDTA. [3aP]DNA was prepared from phages purified by iaopycnic centrifugation in CsCl as described before (Holloman et al., 1975).

(c) Preparation

of fragments

Random fragments of single-stranded +X174 DNA were produced by digestion with pancreatic DNAase ae described by Holloman et al. (1975). Their average length was estimated ae described before (Holloman et al., 1975) or by their rate of sedimentation in alkaline sucrose relative to intact strands. Specific fragments were produced by cleavage of single-stranded $X174 or G4 DNA with endonucleaae R *Hue111 (Blakesley & Wells, 1975; Horiuchi t Zinder, 1976) aa described previously (Beattie et aE., 1977). The reisolated specific fragments were 60 to 90% pure by mass, aa determined by a second electrophoresis on polyacrylamide gels. t See footnote

on page 805.

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These estimates were made from densitometer tracings of autoradiograms of the gels, Nearly all of the contamination of any reisolated fragment was due to smaller fragments. The extent of contamination was therefore even larger in terms of relative numbers of fragments. The sizes of purified specific fragments were determined by comparison of their electrophoretic mobilities to the mobilities of fragments of #X174 or G4 RF DNA of known size in polyacrylamide gels containing 99% formamide (Maniatis et al., 1975; Beattie et al.. 1977). (d) Porrnation and purification of complexes Complexes were formed by incubation of specific homologous fragments with $X174 RF1 or G4 RF1 for 2 to 3 h at 75°C in 0.2 M-NaCl, 10 mM-Tris*HCl (pH 7*5), 1 mM-EDTA at a ratio of 1 or 2 fragments/molecule of RFI. Complexes containing random homologous et al., 1975). Complexes prepared fragments were prepared as described before (Holloman with small fragments (I < 100) were separated from unassociated fragments either by sedimentation through neutral sucrose or by gel filtration on a 0.3 cma x 20 cm column of Sepharose 2B in 0.1 M-NaCl, 1 mM-Tris.HCl (pH 7*5), 1 mM-EDTA. In each case the fractions that contained [3H]RFI were pooled, precipitated overnight with 2.5 vol. ethanol at -20°C and redissolved in 20 mM-NaCl, 1 mn/l-Tris.HCl (pH 7=5), 1 m&IEDTA. Complexes containing large fragments (I > 100 nucleotides) were separated from IWII and unassociated fragments by centrifugation for 48 h at 43,000 revs/min in a Beckman type 50 rotor in a solution of CsCl, p = 1.580, and 100 to 200 rg ethidium bromide/ml. The DNA in the position of form I was pooled, extracted 4 times with redistilled isoamyl alcohol to remove the ethidium bromide, and dialyzed against 20 m&t-NaCl, 1 mM-Tris.HCl (pH 7.5), 1 mM-EDTA. In one experiment (see Fig. 3), complexes were eluted from a nitrocellulose filter by soaking it in 1 mM-Tris*HCl (pH 7.5), 1 mM-EDTA for 12 h at room temperature. Complexes were assayed as described by Beattie et al. (1977) and characterized as described i11 the legend t,o Table 1.

(e) Sucrose gradient sedimentation Sedimentation through gradients of neutral sucrose was carried out in linear gradients of 5% to 20% sucrose in 1 M-NaCl, 10 mM-Tris*HCl (pH 7*5), 10 mM-EDTA. Alkaline sucrose contained 5% to 20% sucrose in 0.7 M-NaCl, 0.3 M-NaOH, and 10 mM-EDTA. All sucrose gradients were centrifuged at 4°C in cellulose nitrate tubes. In all of the diagrams shown here the direction of sedimentation was from right to left. (f) Determination

of radioactivity

Samples (0.01 to 0.2 ml) from gradients of sucrose or CsCl, and from chromatography columns were diluted with 0.7 ml water and dissolved in 5 ml of a solution containing per liter: 600 ml toluene, 358 ml Triton Xl00 (New England Nuclear) and 42 ml Liquifluor (New England Nuclear). Nitrocellulose filters were dried and placed in 3 ml of Econofluor (New England Nuclear). Radioactivity was determined in a liquid scintillation counter.

(g) Electron microscopy DNA was prepared for electron microscopy by the modification of the basic proteilh film technique described by Bastia et al. (1975). Samples which contained 50% formamide were spread over water, picked up onto Formvar film backed with carbon, stained in many1 chloride and shadowed with platinum-palladium at an angle of 10” on a rotating platform. Molecules in random areas from each of several grids were photographed and counted. Contour lengths of the DNA were determined by using a map measurer on enlarged tracings of the molecules.

3. Results (a) Base-pairing Previous observations taken up by superhelical

of the single strand that is taken up by superhelical have indicated that DNA is base-paired

DNA

some part of the single strand that is (Holloman et al., 1975; Beattie et al.,

808

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C. WIEGAND

ET

AL.

1977). The base-paired portion of the fragment should be resistant to digestion by nucleases that act specifically on single strands, such as exonuclease VII of E. coli, which initiates digestion from either the 3’ or the 5’-end (Chase & Richardson, 1974b). When purified complexes that contained specific 32P-labeled fragments 285 to 1200 nucleotides in length (Table 1 and Figs 1 and 2) were digested with sufficient exonuclease VII to have made all of the single-stranded DNA acid-soluble, a core of fragment of average length 300 nucleotides (Table 1) was resistant to digestion in all cases, as judged by the amount of 32P that sedimented with the double-stranded [3H]DNA (Fig. 2). The lowest concentration of enzyme removed all but about 300 nucleotides from the fragments which initially had 1020 to 610 nucleotides. The enzyme digested few, if any, nucleotides from the fragment which was 320 nucleotides long (Fig. 2(a)). The fragments remaining in the complexes were relatively resistant to further digestion. Higher concentrations of enzyme removed another 50 to 100 nucleotJides from the fragment (Fig. 2(a)). Similar results were obtained when complexes of G4 DNA containing fragments of 250 or 690 nucleotides were digested for varying amounts of time (Fig. 2(b)). E xonuclease VII removed few nucleotides from the fragment which was 250 nucleotides long, but rapidly trimmed the larger fragment to a residuum of about 325 nucleotides. In each case the percentage of RF present as complexes remained constant. Other nucleases also digest only part of the single strand taken up by superhelical DNA. When complexes which contained an average of 254 nucleotides of fragment per RF were extensively digested with S, nuclease about 170 nucleotides per RF were TABLE

Complexes of +X174 RFI Fragment

Length

DNA and speci$c single-stranded fragmentst

(nucleotides)

Hue-A’

1200

1

%RF in complext

24

i,§

1004

i, after exoVI1 286

Hae-B’

1020

42

860

322

Hae-D’

610

67

430

280

Hae-E’

320

51

330

333

Complexes made as described in Materials and Methods were purified by sedimentation through gradients of neutral sucrose for 14 h at 41,000 revs/min in a Beckman SW41 Ti rotor followed by centrifugation in CsCl in the presence of 200 pg ethidium bromide/ml as described in Materials and Methods. The [32P]fragments had 2.7 x lo7 cts/min per pmol, the RF1 [3H]DNA used in the preparations made with fragments Hue-A’ and Ha.e-B’ had 2.3 x lo7 cts/min per pmol, and the RF1 [3H]DNA used in the preparation made with fragment Hae-D’ and Hae-E’ had 3.86 x 10’ cts/min per pmol. The average number of nucleotide residues of fragment DNA per molecule of RF1 (P) was ascertained from the amount of [32P]DNA in the position of RF in neutral sucrose gradients (Fig. 1). The number of residues of fragment per molecule of complex (rO) was calculated by dividing i by the fraction of the RF which had taken up fragments. The latter was determined by the extent of binding of 3H to nitrocellulose filters as described by Beattie et al. (1977). The values of rc after digestion of the complexes by exonuclease VII (exoVI1) were calculated from the data shown in Fig. 1. t Fragments produced by digestion of single-stranded DNA with endonuclease R . Has111 are designated Hae-A’, Hue-B’ . . . Hae-N’ in order of decreasing size, by analogy with the nomenclature of Smith & Nathens (1974) for double-stranded fragments. .$ Taken as equal to the %RF retained by a nitrocellulose filter (see Beattie et al., 1977). 5 Average number of nuoleotide residues of fragment per molecule of complex.

0

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R

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R. C. WIEGAND

810

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ET

AL.

I

I

I

I

I

I

2

4

0

20

40

6C

Units/ml

Time (min)

(a)

(b)

FIQ. 2. Resistance of part of the third strand to exonuclease VII. (a) Complexes, which are described in Table 1, were digested with 1.4, 2.8 or 6.5 units of exonuclease VII/ml in 0.2-ml reaction mixtures containing 60 mM-potassium phosphate (pH 7.6), 26 mM-potassium EDTA, 0.6 mi\l-dithiothreitol and 13.6 PM-DNA complexes. After incubation at 37°C for 45 min the reaction mixtures were chilled, laid onto a gradient of neutral sucrose and centrifuged 4 h at 60,000 revs/min in a Beckman SWSO. 1 rotor. The number of residues per oomplex was calculated from the percentage of RF molecules that had formed complexes and the specific activities of the DNA (Tahle 1). ---m--, Complexes made with Hue-B’ (I = 1020); -n--n-, Hoe-D’ (I = 610); ---A--, Hue-E’ (I = 320). (b) Complexes of phage G4 RF and specific single-stranded fragments were formed by incubating 70 nmol of fragment with 200 to 400 nmol of RF1 in 0.6 ml of 0.2 M-NaCI, 10 mnr-Tris*HCl (pH 7.5), 1 mM-sodium EDTA. After 3 h at 76”C, a third of the RF had formed complexes. The complexes were then incubated at 37°C in 3.2 ml of a mixture containing 2.2 units of exonuclease VII/ml, 60 mM-potassium phosphate (pH 7.9), 13 mM-potassium EDTA, 6 mm-dithiothreitol and 60 to 120 q-complexes. After 0, 10,20, 30,40 and 60 min at 37”C, samples of 0.6 ml were removed to tubes on ice containing 0.26 ml 6 M-NaCl and 0.26 ml 1 M-sodium acetate (pH 6). Samples were centrifuged through gradients of neutral sucrose for 22 h at 27,000 revs/min in a Beckman SW27.1 rotor. The number of residues per complex was calculated as in (a). -O-O-, Complexes made with Hae-C’ (2 = 690); -O-O--, Hue-F’ and G’ (1 = 260) (see Beattie et al., 1977).

resistant (see Fig. 6). Some of the fragment nucleotides were also resistant to digestion by the recBC nuclease (see Fig. 9). Most complexes prepared with fragments large enough to unwind all or nearly all of the superhelical turns sediment more slowly than RF1 in neutral sucrose (Beattie et al., 1977; and Fig. l(a) and (b)). I n each of the preparations shown in Figure l(a) and (b) the major peak of [32P]fragment sedimented more slowly than the leading peak of 3H, which marks the position of RFI. In addition, some of the 32P sedimented more rapidly than the major peak. This may have been due in part to contamination of the larger purified restriction fragments with smaller fragments (see Materials and Methods). All of the complexes made with a fragment of 320 nucleotides sedimented more rapidly, in the position of form I (Fig. 1 (c)). Following digestion with exonuclease VII all of the [3H]RF and [32P]fragment sedimented in the position of RFI, without regard to the original size of the fragment (Fig. l(d) to (f)). The sedimentation rate of complexes made uith large fragments increased following digestion with exonuclease VII, which probably did not result from digestion of paired bases, since our preparation of exonuclease VII had no detectable activity on double-stranded DNA (see Materials and Methods). Rather, the increased rate of sedimentation can be attributed

SUPERHELICAL

DNA

UPTAKE

OF FRAGMENTS,

x11

III

t,o the differences in temperature and ionic strength of the reaction mixture versus t,he sucro.
IOOl

J./-

X

so ‘D : 0 F

,I s

I x’

. 60-

1’

_

X -

40 20

. /

-

/

/

X/IX / /’ AX

0

I

I

I

I

55

65

75

85

.-d--x* 055 Temp.

(a)

I 65

75

85

I _ 95

PC ) ib)

FIG. 3. Melting curves for complexes and RFII. (a) Thermal stability of complexes of 4X174 RF1 DNA and homologous random fragments t,he average length of which was 1000 nucleotides. The solutions, which contained 1 mM-Tris*HCl (pH 7.5), 0.1 mM-EDTA, and either 20 mM or 0.5 M-NaCl, were heated and kept at the temperatures indicated for 5 min prior to sampling. Samples of 0.02 ml containing 0.3 nmol of [3H]RF nucleot,ides were added to 2.4 ml of cold 10 x SSC and filtered through nitrocellulose as described by Beattie et al. (1977). (b) Stability of $X174 RF11 under the same conditions. RF11 was prepared as described previously (Wiegand et al., 1975). -@---a--, 20 m&I-NaCl; --x --x --, 0.5 M-NaCl. The complexes that were melted in 20 m&r-NaCl had been purified by adsorption to a nitrocellulose filter followed by elution and centrifugation in C&l and ethidium bromide (see AMaterials and Met,hods).

The thermal stability of complexes is also consistent with base-pairing of the fragment. When complexes were heated in either 20 mM or 0.5 M-NaCl the t, of the complexes was similar to that of $X174 form II DNA under the same conditions (Fig. 3). The melting curves of complexes were sharper than those for form II, as might be expected because the fragments are smaller. The thermal stability of complexes was perplexing when compared to their apparent instability on exposure to ethidium bromide (Holloman et al., 1975). We therefore reinvestigated the stability of complexes in ethidium bromide. Complexes made with small fragments (I < 100) were dissociated by ethidium bromide (Holloman et al., 1975). Complexes made with larger fragments (I > 280) were stable to 300 pg ethidium bromide per ml for prolonged periods (Pig. 4). (b) Electron microscopic examination of complexes A preparation of complexes made with a fragment 1020 nucleotides long was examined by electron microscopy. The DNA was spread in 50% formamide in order to extend any single-stranded DNA which was present. The conditions used made it ,x

R. C. WIEGAND

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AL.

(0)

4(

3c

2c

n-

IC

‘0 x z \ u1 t

C

(b)

,I

IC

5

Froctlons

Fra. 4. Stability of complexes in CsCl and ethidium bromide. (a) RF1 [3H]DNA and fragment [eaP]DNA (I = 2500) were added to a solution of CsCl (p = 1,680) and ethidium bromide (300 &ml). (b) Complexes were prepared with random fragments (I = 2600, i = 1300) and purified from unassociated fragments by chromatography on Sepharose 2B (see Materials and Methods). CsCl was added to the complexes to make the density 1.680 and ethidium bromide was added to 300 pg/ml. The solutions were centrifuged for 48 h at 43,000 revs/min in a Beckman type 60 rotor at 20°C. Fractions were collected from the bottoms of the tubes and samples counted. According to the 3H and 32P in fractions 21 to 29 of(b) the peak of more dense material contained about 1300 residues of [32P]nucleotide per molecule of RF. -a--•--, [3H]DNA; --x --x --, [32P]fragments.

unlikely that the difference in thickness between double-stranded and single-stranded DNA could be detected. Among 105 molecules observed from the preparation of complexes, 26 (25%) contained bubbles (Fig. 5(a)). Among 72 molecules observed from the RFI, none contained bubbles (Fig. 5(b)). U n d er similar conditions of preparation for microscopy the contour length of single-stranded DNA is nearly the same as that of double-stranded DNA (Freifelder et al., 1964; Chandler et al., 1964). Each arm of the observed bubbles in our complexes was about 6% of the total contour length which equals 324 nucleotides or nucleotide pairs. This number is consistent with the length of the paired region determined by the enzymological approach just described. The formation of 324 base-pairs between the fragment and the +X174 form

SUPERHELICAL

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FRAGMENTS,

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813

I DNA should have removed many of the superhelical turns. Although electron microscopic visuaIization of DNA does not give a reliable estimate of the superhelix density it is interesting to note that, whereas all of the RF1 molecules appeared twisted, none of the molecules that contained a loop appeared twisted. If 324 nucleotides were paired, a single-stranded tail of 696 nucleotides should have been visible in the molecules which had loops. Such tails were observed in only seven molecules (27%) of the 26 which contained loops. Tails may not have been visible due to their small size or some artifact of preparation. Since preparations of specific fragmrnts tend to be contaminated with shorter fragments (see Materials and Methods) it. may be that the tails in some of the complexes were shorter and more difficult to we. (c) Cleavage of displucement loops (D-loops) by & nwlease The uptake and base-pairing of a third strand by duplex DNA implies either that all three strands are in some hydrogen-bonded structure (Lacks, 1966) or that one strand of the recipient molecule is unpaired, as in the D-loop of mitochondrial DNA (Kasamatsu et aE., 1971). The following data, together with other evidence (Holloman et al., 1975; Beattie et al., 1977), demonstrate that uptake of a third strand by superhelical DNA produces a D-loop. Digestion of complexes for two hours at 4°C with S, nuclease as described in the legend to Figure 6 converted all of the form I [3H]DNA to form II. Under those conditions, 67% of the [32P]fragment present in the complexes was retained in the purified cleaved complexes, about 170 nucleotide residues of fragment per molecule of RF (Fig. 6(e)). The efficiency of retention of fragment in cleaved complexes was much greater when the incubation with S, was at 4°C than it was at 37°C (Holloman et al., 1975). When the complexes that had been cleaved by S, were heated for one minute at 60°C in 50 mnn-TrisaHCl (pH 7*5), 25 mM-EDTA and 20 mM-NaCl the [32P]fragments remained associated with the form II [3H]DNA (Fig. 6(f)). When similar complexes, containing 254-nucleotide residues of fragment, were cleaved at random with pancreat’ic DNAase at 22°C in 50 mi\l-Tris *HCI (pH 75), 0.5 mM-MgCl,, and 20 mM-NaCl most of the form I [3H]DNA was converted to form II DNA and 537; of the [32P]fragment nucleotides (i’ = 135) remained associated with form 1I (Fig. 6(c)). However, when these nicked complexes were heated for one minute at 6O”C, all of the fragments dissociated from form II DNA (Fig. 6(d)). As a control for the experiments with both S, nuclease and pancreatic DNAase, intact complexes were heated for one minute at 60°C in 10 mM-Tris *HCl (pH 75), 5 mM-MgCl, and 12-5 mM-Nacl. None of the fragments dissociated (Fig. 6(a) and (b)). As before (Holloman et al., 1975) we interpret these observations as indicated in Figure IO(c) and (d) of this paper. Random nicking by pancreatic DNAase releases t,he energy of superhelix formation which results eventually in displacement of the third strand by branch migration (Radding et al., 1977; Robberson & Clayton, 1973). S, nuclease also relaxes the [3H]RF by cleaving it, but since the enzyme digests one strand of the recipient at the site of uptake of the homologous fragment, the latter can no longer be displaced by branch migration. In similar experiments approximately 400 nucleotides of RF [3H]DNA per complex were released as acid-soluble counts by S, digestion of complexes made with specific fragments. This is close to the amount of fragment DNA shown to be base-paired in complexes (see Table 1, Figs 1 and 2).

814

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(d) Recombinant molecules from joint molecules Our interpretation of the action of S, nuclease is supported by the following experiment. Complexes prepared with a specific fragment 1020 nucleotides in length were cleaved with S, nuclease as described above. The cleaved complexes had an average of 115 residues of fragment nucleotide per molecule of RF (data not shown). The cleaved complexes were incubated with an excess of polynucleotide ligase and the product was analyzed by sedimentation in alkaline sucrose (Fig. 7(b)). We observed the linkage of 43 [32P]nucleotide residues per molecule of RF. This is 37o/o of the number of residues associated with complexes after digestion by S,. We did not detect complexes that had been sealed at both ends of the fragment to yield a closed

SUPERHELICAL

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UPTAKE

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515

(b) FIG. 5. Electron 0.5 pm.

micropraphs

of (a) complexes

and (b) $X174

RF1 I)NA.

Thr~ bar wprc

:sPnts

circular molecule containing 32P. Since polynucleotide ligase linked one end of 37” I 0 of [““P]l ?ragments to [3H]strands, on a random basis ligase might have linked both ends of 14% of fragments. In the experiment shoun in Figure 7 this would have resulted in about 40 cts/min of 32P in the position of covalently closed circular I )NA, which u rould have been barely detectable. Most of the closed circular molecul es of [ 3H]DN. A observed in Figure 7 were not due to the closure of complexes, but ri sther to the c:losure of unassociated form IT DNA which was present in the digest ISC?C Wieganc 1 et al., 1975).

R. C. WIEGAND

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(b) I3.4

20

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30

FIG. 6. Nicking of complexes weraua digestion of the displacement loop: effects on the association of the third strand. Complexes were made as described before (Holloman et al., 1975) and separated from free single-stranded fragments by sedimentation through neutral sucrose for 18 h at 30,000 revs/min in a Beckman SW41 Ti rotor ((a) and (b)), or by gel filtration ((c) to (f), see Materials and Methods). After the indicated treatments, samples were laid on gradients of neutral sucrose and centrifuged for 3 h at 60,000 revs/min in the Spinco SW50.1 rotor. All samples in the right-hand frames were -@---a-, 3H originally in 4X174 RF; heated at 60°C for 1 min before centrifugation. --x --x --, e2P in fragments of 4X174 viral strands. The average length in nucleotides of fragments which were produced by digestion with pancreatic DNAase was 63 for the complexes used in (a) and (b) and 50 for the complexes used in (c) to (f)). (a) Control: untreated complexes; i = 93. (b) The same complexes heated for 1 min at 60°C in 10 mnr-Tris,HCl (pH 7.6), 5 mM-MgCl,, and 12.6 mlur-NaC1; i = 109. (c) to (f) The control value of i: for the complexes used in these experiments was 264 nucleotide residues per molecule of RF. (c) Pancreatic DNAase: a mixture (0.1 ml) containing 50 rnx-TrisbHCl (pH 7.6), 20 m&r-NaCl, 0.6 m&r-MgCl,, 63 ~M-[~H]DNA nucleotide and 10 ng pancreatic DNAase/ml was incubated for 3 min at 22°C. The reaction was stopped by the addition of 10 ~1 of 0.5 M-EDTA. For the material in the major peak of DNA, i was 136, which was 53% of the control value. (d) A sample as in (c) heated at 60°C for 1 min after the addition of EDTA. (e) S1 nuclease: a mixture (0.3 ml) containing 50 mm-sodium acetate (pH 5), 0.1 &r-NaC1, 1 mp/r-ZnSO,, 426 PM-DNA nucleotide and 210 units of Si nuclease was incubated for 1 h at 4°C at which time an additional 210 units of enzyme were added. After another h at 4”C, the complexes were purified by filtration through Sepharose 2B (see Materials and Methods). DNA in the peak fractions was precipitated by ethanol (see Materials and Methods) and redissolved in 0.1 ml of 0.1 nil-NaCl, 1 mM-Tris.HCl (pH 7.5) and 1 mM-EDTA. i = 170 which was 67% of the untreated control. (f) A sample of the material described in (e) was heated at 60°C for 1 min in 60 m&rTris,HCI (pH 7.5), 26 mM-EDTA and 20 mM-NaCl; P = 177.

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,I 4

20

30

Fractions

Fm. 7. Covalent linkage of fragments of RF in &-treated complexes. Complexes were prepared as described in TabIe 1 from RF1 r3H]DNA having 2.5 x 10’ cts/min per pmol and fragment Hae-l3’ (I = 1020 nucleotides) [32P]DNA which had 1.73 x lOa cts/min per pmol. The preparation of complexes had on the average 240 nucleotides of fragment per molecule of RF. Complexes (120 nmol) were digested with 90 units of S1 nuclease in 0.2 ml of 50 m&l-sodium acetate (pH 6), O-1 ~-N&cl and 1 mM-.&SO, for 1 hat 4°C. The reaction was stopped by addition of potassium phosphate (pH 7.6) to 10 mM and the DNA was precipitated by addit,ion of 2.5 vol. cold ethanol. The preparation of cleaved complexes, which were resuspended in 1 mMTris.HCl (pH 7.5), 1 mM-EDTA and 0.196 ethanol, contained on the average 115 residues of fragment/RF. The cleaved complexes were incubated (a) without or (b) with 1 unit of E. coli polynucleotide ligase in a reaction mixture containing 30 mM-Tris.HCl (pH S), 4 mnr-MgCl,, 1.2 miw-potassium EDTA, 1.7 mM-%mercaptoethanol, 26 FM-j-diphosphopyridine nucleot,ide, 0.05 mg bovine serum albumin/ml and 13 @-cleaved complex (as nucleotide in RF). After 30 min at 37°C EDTA was added to 50 mM and the samples were centrifuged 2.5 h at 50,000 revs/min in a Beckman SW50.1 rotor through gradients of alkaline sucrose. -e---a-, 3H cts/min; --x --x --, aaP cts/min.

Covalent linkage of the fragment to a strand of the recipient RF by the sequential action of S, nuclease and polynucleotide Iigase proves that S, n&ease cleaved a st’rand of the recipient DNA at the site of uptake as indicated in Figure IO(d). (e) Cleavage of D-loops by recBC DNAase

The recBC DNAase of E. c&i, which has been implicat’ed in recombination (Clark, 1974; Tomizawa & Ogawa, 1972), has properties that are suited to a role in cleaving

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D-loops. The enzyme degrades single-stranded DNA both endonucleolytically and exonucleolytically (Goldmark & Linn, 1972), it degrades double-stranded DNA exonucleolytically, but will not initiate digestion at a nick (Wright et al., 1971; Karu et al., 1973), it has no endonucleolytic action on relaxed double-stranded DNA (Karu et al., 1973), or on superhelical DNA (Goldmark & Linn, 1972) as illustrated by the control experiment shown in Figure 8.

FIQ. 8. The recBC nucleeee does not cleave 4X174 RF1 DNA. (a) +X174 RF1 DNA (6 nmol) was sedimented 2 h at 60,000 revs/min through a gradient of alkaline sucrose in a Beckman SW60.1 rotor. (b) +X174 RF1 DNA (6 nmol) was incubsted for 46 min at 37°C with 6.8 units of recBC nuclease in 0.1 ml of 50 mM-Tris.HCl (pH 8.5), 10 rnMMgCI,, 0.67 mm-dithiothreitol, O-1 m&f-ATP and 1 mg acetylated bovine serum albumin/ml. The reaction was stopped by addition of EDTA to 50 rnM and the sample was centrifuged as in (a).

Complexes made with large fragments sedimented more slowly in neutral sucrose than RFI, overlapping substantially with RF11 (Fig. 9(a), also see Fig. 1 and Beattie et al., 1977). In alkaline sucrose most of the 3H in these complexes sedimented in the position expected for closed circular DNA (Fig. 9(c)). After digestion by recBC DNAase most of the 3H sedimented in two peaks in neutral sucrose, the leading one in the position expected for form I DNA, the trailing one in the position expected for form II DNA. All of the 3H in the second peak (Fig. 9(b)) was in form II molecules as shown by its slow sedimentation in alkaline sucrose (Fig. 9(d)). According to the amount of 3H left in the leading peak of Figure 9(b) the recBC DNAase cleaved about half of the form I DNA in the preparation of complexes. Since 69% of the form I DNA was present as complexes, the recBC enzyme cleaved 72% of the complexes. The cleavage of complexes was stimulated by ATP. Maximal digestion of [32P]strands and cutting of [3H]RF occurred at 0.1 mM-ATP. The activity was lower by at

SUPERHELICAL

DNA

UPTAKE

20

OF

FRAGMENTS,

81!)

111

_(b)

1 IO ,

(d)

.A. I\

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/b

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30

Fractions FIG. 9. Cleavage of complexes by the recBC DNAase of E. coli. An endo R.HaeIII fragment of 4X174 [32P]DNA (2.7 x lOa cts/min per pmol) 610 nucleotides long was prepared and purified as described in Materials and Methods. Complexes were prepared using +X174 RF1 [3H]DNA (2.6 x 10’ cts/min per pmol). The complexes were purified as described in Materials and Methods by centrifugation in CsCl/ethidium bromide. The separation of complex from unassociated fragments was not complete in this purification (a). About 20% of the molecules in th(x purified complexes were form II (c). Complexes (5 nmol) were incubated (a) without or (b) with 10 units of recBC DNAase in 0.1 ml of 50 max.Tris.HCl (pH 8.6), 10 mM-MgCl,, 0.67 mnr-dithiothreitol, 0.1 miw-ATP and 1 mg acetylated bovine serum albumin/ml. After 46 min at 37”C, the reactions were stopped by addition of EDTA to 50 mM, and the samples were centrifuged 4 h at 50,000 revs/min through gradients of neutral sucrose in a Beckman SWKO.l rotor. (c) Complexes (1 nmol) were incubated as above without recBC nuclease. After 45 min at 37”C, EDTA was added to 50 mM and the sample was centrifuged 2 h at 60,000 revs/min through a gradient of alkaline sucrose in a Beckman SW60.1 rotor. (d) A similar preparation of complexes w&s digested with recBC nuclease as described above. After 45 min EDTA was added to 60 mM to stop the reaction and the sample was centrifuged through a gradient of neutral sucrose for 18 h at 27,000 revs/min in a Beckman SW27.1 rotor. Fractions were collected and a sample from each was counted. Fractions in the normal position of form II which contained radioactive DNA were pooled and the DNA was precipitated with 2.5 vol. et,hanol overnight at - 20°C. The precipitated DNA was redissolved in 20 mM-NaCl, 10 mMTrin*HCl (pH 7.5), 1 mM-EDTA and centrifuged 2 h at 60,000 revs/min through a gradient of alkaline sucrose in a Beckman SW50.1 rotor. Radioactivity due to the dissociated [3zP]fragments was not plotted for (c) and (d). -a-a-, SH ct,s/min; --x --x --, azP cts/min.

least a factor of two at either 1 mM or 10 PM-ATP. There was little, if any, digestion of [32P]strands or L3H]RF in the absence of ATP. The recBC DNAase digested some of the 32P-labeled strand in complexes. Most of the undigested [32P]strand was associated with form II [3H]DNA (Fig. 9(b)). To calculate the number of residues of [32P]nucleotide remaining per molecule of cleaved complex, it was necessary to take account of the small amount of relaxed [3H]RF that was present before digestion with recBC DNAase (Fig. 9(c)). A count by electron microscopy showed that linear molecules comprised only 1% of molecules in either the preparation of RF1 or complexes. Therefore we assumed that the relaxed DNA present before digestion was form II and was not further degraded by the recBC

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DNAase. Accordingly an appropriate subtraction was made from the total amount of form II E3H]DNA present after digestion. In two experiments, in each of which the third strand was 310, 610 or 1020 nucleotides, digestion by recBC DNA&se left 215 & 43 residues of [32P]nucleotide per molecule of RF. This value is consistent with the observations of the action of exonuclease VII and S, nuclease. Furthermore, as in the experiments on S, nuclease (Fig. 6), the [32P]DNA which remained associated with form II [3H]DNA was not displaced when the digest was heated at 60°C for one minute in 50 mM-Tris=HCl (pH 7*5), 10 mM-EDTA and 20 m&r-NaCl (data not shown). As in the experiments on S, nuclease, we interpret the latter observation to mean that form I [3H]DNA in the complexes was converted to form II DNA by digestion of the displacement loop at the site of uptake of the [32P]strand (Fig. 10).

s, or recec nuclease

FIG. 10. Interpretation of experiments on the formetion and cleavage of complexes. (a) Str8nd uptake produces 8 displacement loop and unwinds the super-helix. If the fragment is large enough to unwind all or nearly all of the superhelical turns the RF will sediment 8s a relaxed form, slower then the native superhelical form. (b) Cleavage of the complex with exonuclease VII (exoVI1) removes 8ny single-stranded ends. (c) Random but limited cleavage of the RF by pencrestic DNAase (Pant DNese) allows the strands of the RF to rotate freely and displace the fragment by bmnch migration. (d) Cleavage of the D-loop by single-strand-specific endonucleases stabilizes the association of the donor strend. (e) Polynucleotide ligate covalently links the donor strand to 8 strand of the recipient RF molecule after cleavage of the complex by Si nuclease.

Since the endonucleolytic activity of the recBC DNAase works only on singlestranded DNA, the enzyme might cleave D-loops simply by recognizing the displaced single strand. To study the specificity of the enzymic action on D-loops, we mixed 3.4 nmol (expressed as nucleotide residues) of single-stranded circular +X174 [32P]DNA with an equal amount of a preparation of complexes in which 56% of [3H]RFI molecules had taken up a third strand of average length 1000 nucleotides. Thus the mixture included 4.1 x 101’ molecules of single-stranded circular DNA and 1.14 x 1O’l molecules of double-stranded circular DNA that contained D-loops. From the patterns of sedimentation in neutral sucrose we measured the disappearance of single-stranded circular [32P]DNA and superhelical [3H]DNA, after digestion by

SUPERHELICAL

DNA

UPTAKE

OF FRAGMENTS,

III

x21

recBC’ DNAase. In 45 minutes, 6.8 units of recBC DNAase made at least one cut per molecule in 2.4 x 1011 single-stranded circular molecules and in 5.42 x IO’O molecules of complexes. Thus the same proportion of the two substrates was cleaved, which shows that the enzyme recognized complexes at least as well as it recognized singlest,randed circles. This is noteworthy because the single-stranded displacement loop in a molecule of complex is only about l/18 of the length of a single-stranded circle. These observations suggest that the cleavage of D-loops is more specific by an order of magnitude than endonucleolytic action of recBC DNAase on single-stranded DNA. Similarly when 2.28 x 1011molecules of complexes were incubated alone with 6.8 units of enzyme, 1.58 x 1011 molecules were cleaved; when 8.2 x 1O’l molecules of singlestranded circular DNA were incubated alone with 6.8 units of enzyme, 2.85 x 101’ molecules were cleaved. These data also support the conclusion that the enzyme cleaved complexes more rapidly than expected on the basis of the single-stranded DNA in the D-loop. Other features of the complex that the enzyme may have recognized include the single-stranded tail of the third strand, the branched structure of t,he D-loop, or the double-stranded portions of the molecule. There are several other ways in which the action of the recBC DNAase is more complicated than expected for simple digestion of the single-stranded displacement loop. First, polynucleotide ligase failed to link the remaining [32P]strand to the cleaved [3H]strand. Several obvious explanations for the failure of ligase to seal digested complexes were tested. The recBC DNAase may have left undigested single-stranded tails. To test this possibility complexes that had been cleaved by recBC DNAase were digested with S, nuclease prior to the incubation with ligase, but there was still no detectable linkage of [32P]fragments to [3H]strands. In a parallel reaction that served as a control, previously untreated complexes were digested with S, nuclea,se and then incubated with ligase, in which case about 37% of the [32P]fragments were covalently linked to [3H]RF strands (Fig. 7(b)). recBC DNAase probably did not leave gaps at the ends of the digested loops either. Complexes that had been digested sequentially with recBC DNAase and S, nuclease were not linear molecules as judged by their resistance to further digestion by phage X exonuclease. Had gaps been present in the complexes digested by recBC DNAase further digestion by S, nuclease should have produced linear molecules, but the experiment may have failed to detect a gap of one or a few nucleotides. The possibility that under these conditions the recBC DNAase left other than the 3’ hydroxyl and 5’ phosphoryl ends required by ligase has not been investigated. The second perplexing feature of the action of recBC DNAase on complexes is shown in Figure 9(b). In spite of the presence of only 1 y0 of linear molecules of DNA in the preparation of complexes (see above), 20% of the [3H]RF was digested to small products that remained near the top of a neutral sucrose gradient (Fig. 9(b)) (see Discussion).

4. Discussion These experiments show that uptake of a single-stranded fragment by superhelical DNA in vitro produces a D-loop similar to that first observed in mitochondrial DNA (Kasamatsu et al., 1971). The third strand can form about 300 base-pairs with the complementary strand of +X174 RFI. When the third strand was 320 nucleotides long, all of its bases were paired (Fig. 3). When the third strand was longer, excess nucleotides were located in single-stranded tails that were susceptible to digestion by

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exonuclease VII. Thus, under the conditions of digestion by exonuclease VII (see legend to Fig. 1) there were at least 30 superhelical turns in $X174 RFI, assuming one turn of the helix for every ten base-pairs. Under the conditions of preparation for electron microscopy, there were also some 300 bases paired in the D-loop. At 4°C in 1 M-NaCl there was evidence of additional superhelical turns that could be removed by the formation of base-pairs with longer fragments (Fig. 1). For RF1 of the related phage G4, other experiments put an upper limit of 40 on the number of superhelical turns in 0.01 M-NE&I at 4°C (see Fig. 5 in Beattie et d., 1977). While a precise comparison is not possible, these values for the number of superhelical turns in 4X174 RF are reasonable in relation to the superhelix density determined in ethidium bromide and CsCl (Wang, 1969b,1974). Further support for our general view of the structure produced by strand uptake comes from observations made by electron microscopy, and observations on the digestion of the displacement loop by endonucleases (see below). There also appear to be structural features that we do not understand. The recBC DNAase acts on doublestranded DNA only exonucleolytically and will not initiate such digestion at a nick (Wright et al., 1971; Karu et al., 1973), yet a significant fraction of [3H]RF from complexes was made acid-soluble by highly purified enzyme. Possibly secondary structure in the displacement loop, or in the tail of the donor fragment, produced a double-stranded end that enabled the recBC DNAase to initiate exonucleolytic digestion. Other evidence of such secondary structure will be described in the following paper (Radding et al., 1977). A complex related to the one that we have described here was reported by Liu & Wang (1975). It consisted of closed circular DNA of phage PM2 with loops, both arms of which were double-stranded. As reported above, the formation of base-pairs between single-stranded fragments and superhelical DNA removed superhelical turns. The number of bases paired in the two studies had a similar relationship to the superhelical density of the circular DNA. According to a recurrent hypothesis in the literature, genetic recombination commonly starts by the interaction of a single strand from one molecule with the duplex DNA of another (Fig. 11 and Hotchkiss 1974; for other citations see Holloman $ Radding, 1976). We have used the uptake of single strands by superhelical DNA as a simple experimental system to explore that idea. Two features of strand uptake might, in large part, determine the initiation of recombination. These are the driving force of the energy of superhelix formation (Holloman & Raclding 1976 ; Beattie et al ., 1977) and the stereochemistry of the product. Strand uptake produces a particular kind of joint molecule, in which an intact strand of a recipient molecule is unpaired as a result of displacement by an identical donor strand. Recombination by breakage and reunion requires that both parental molecules be cut in about the same place. The structure of a D-loop, formed as an early step in recombination, could determine the location of the cut in the second parental molecule. In vitro, the displaced strand of the recipient molecule is susceptible to cleavage by several enclonucleases that act on single-stranded DNA, including the recBC DNAase of E. coli which has been implicated in genetic recombination (Clark, 1974). Our dat.a suggest in fact that the recBC nuclease cleaves D-loops more specifically than it cleaves single-stranded DNA. The sequence in vitro comprised of strand uptake, loop cleavage, and covalent union simulates the formation of a joint molecule and its conversion into a recombinant molecule.

SUPERHELICAL

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UPTAKE

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Strand uptake

1 \

FRAGMENTS,

III

82.3

I

LOOP cleavage

-1

-7

(b) (a ) FIG. 11. Initiation of genetic recombination. (a) Shows a hypothetical sequence in which strand displacement by a polymerase produces a redundant strand which is taken up by a homologous molecule. Uptake of a donor strand results in cleavage of the recipient molecule, opening it to propagation of a strand transfer. The model proposed by Meselson & Radding (1976) relates the structure on the lower left to reciprocal exchanges. (b) Represents experiments (Holloman et al., 1976; Beattie et al., 1977 and this paper) that partially simulate the hypothetical sequence, and may also be seen as a model for the kind of non-reciprocal recombination that occurs in bacterial transformation (Fox, 1966).

This hypothesis is consistent with the observations of Benbow et al. (1975) on recombination in 4X174 made in vivo. Our finding of uptake of single-stranded DNA driven by the free energy of superhelix formation provides a basis for understanding t,he phenomenon they termed “single-strand aggression.” While the recBC DNAase does not appear to be required for recombination of $X174 (Benbow et al. 1974) or S13 (Tessman, 1968), the hypothetical sequence pictured in Figure 11(a) may be related to the formation of the recombinant “figure 8” intermediate of $X174, S13 and colicin El DNA (Benbow et al., 1975; Thompson et al., 1975. Potter & Dressler, 1976) by mechanisms described by Meselson & Radding (1975). The authors are grateful to Drs D. Eichler and I. R. Lehman for a generous gift of purified recBC DNAase. This research was sponsored by grant no. NPSOB from the American Cancer Society and grant no. CA 16038-02 from the National Cancer Institute. One of us (K.L.B.) was a fellow of the Jane Coffin Childs Memorial Fund for Medical Research. REFERENCES Bast,ia, D., Sueoka, N. & Cox, E. C. (1975). J. Mol. Biol. 98, 305-320. Beattie, K., Wiegand, R. C. & Radding, C. M. (1977). J. Mol. Biol. 116, 7834303. Benbow, R. M., Zuccarelli, A. J., Davis, G. C. & Sinsheimer, R. L. (1974). J. Viral. 13, 898-907. Benbow, R. M., Zuccarelli, A. J. & Sinsheimer, R. L. (1975). 1%~. Nat. .4cud. 8ci., 71.B.A. 72, 235-239. Blakesley, R. W. & Wells, R. D. (1975). Nature (Lo&on), 257, 421-422. Chandler, B., Hayashi, M., Hayashi, M. N. & Spiegelman, S. (1964). Science, 143. 47 49. Chase, J. W. & Richardson, C. C. (1974a). J. Biol. Chem. 249, 4545-4552. Chase, J. W. & Richardson, C. C. (1974b). J. Biol. Chem. 249, 4553-4561. Clark, A. J. (1974). Annu. Rev. Cenet. 7, 67-86.

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Horiuchi, K. & Zinder, N. (1975). Proc. Nut. Acad. Sci., U.S.A. 72, 2555-2558. Hotchkiss, R. D. (1974). Annu. Rev. Microbial. 28, 445-468. Karu, A. E., MacKay, V., Goldmark, P. J. & Linn, S. (1973). J. BioZ. Chem. 248,4874-4884. Kasamatsu, H., Robberson, D. L. & Vinograd, J. (197 1). Proc. Nat. Acad. Sci., U.S.A. 68, 2252-2257. Lacks, S. (1966). Genetics, 53, 207-235. Liu, L. F. & Wang, J. C. (1975). Biochim. Biophys. Actu, 395, 405-412. 14, 3787-3794. Maniatis, T., Jeffrey, A. & van de Sande, H. (1975). Biochemistry, Me&son, M. S. t Radding, C. M. (1975). Proc. Nat. Acad. Sci., U.S.A. 72, 358-361. Middleton, J. H., Edgell, M. H. & Hutchison, C. A., III (1972). J. ViroZ. 10, 42-50. Potter, H. & Dressler, D. (1976). Proc. Nat. Acud. Sci., U.S.A. 73, 3000--3004. Radding, C. M. (1966). J. Mol. BioZ. 18, 235250. Radding, C. M., Beattie, K. L., Holloman, W. K. & Wiegand, R. C. (1977). J. Mol. BioZ. 116, 825-839.

Robberson, D. L. & Clayton, D. A. (1973). J. BioZ. Chem. 248, 4512-4515. Smith, H. 0. & Nathans, D. (1973). J. MOE. BioZ. 81,419-423. Tessman, I., (1968). Science, 161, 481-482. Thompson, B. J., Escarmis, C., Parker, B., Slater, W. C., Doniger, J., Tessman, I. & Warner, R. C. (1975). J. Mol. BioZ. 91, 409-419. Tomizawa, J. & Ogawa, H. (1972). Nature New BioZ. 239, 14-16. Wang, J. C. (1969a). J. Mol. BioZ. 43, 25-39. Wang, J. C. (19698). J. Mol. BioZ. 43, 263-272. Wang, J. C. (1974). J. Mol. BioZ. 89, 783-801. Wiegand, R. C., Godson, G. & Radding, C. M. (1975). J. BioZ. Chem. 250, 8848-8855. Wiegand, R., Beattie, K. & Holloman, W. (1976). Fed. Proc. Fed. Amer. Sot. Exp. BioZ. 3.5, 1594. Wright, M., Buttin, G. & Hurwitz, J. (1971). J. BioZ. Chem. 246, 6543-6555.