’rHE JUURNAL OF BIOLUCICAL CHEMISTRY Vol. 255, No 6, Issue of March 25. pp. 2433-24:3Y, 1980 Prrnted cn ti S A .

Purification and Characterization of the Integration Protein Specified by Bacteriophage A*

(Received for publication, January 29, 1979)

Michael Kotewicz,# Elzbieta Grzesiuk,i?j William Courschesne,n Robert Fischer, and Harrison Echols From the Department of Molecular Biology, University of California, Berkeley, California 94720

The integration (Int) protein of bacteriophage X is required for the insertion of the viral genome into the chromosome of Escherichia coli. Using a DNA-binding assay, we have extensively purified Int protein from two sources: cells infected by wild type phage, and induced lysogenic cells carrying a prophage with the intc mutation that causes constitutive expression of the int gene. The Int protein purified from the two sources appears to be identical; this finding suggests that the intc phage is likely to be the preferred source of wild type Int protein because more Int is produced and there are no regulatory requirements. The most highly puri- fied fraction of Int protein is active in the complete integrative recombination reaction if host factors from uninfected cells are added; thus, Int protein is the only phage-specific protein required for integration. Puri- fied Int has a molecular weight of 40,000, as judged by acrylamide gel electrophoresis of denatured protein; native Int behaves as an apparent monomer during velocity sedimentation in a sucrose gradient. Int pro- tein binds with clear specificity to only one of the two substrates for integrative recombination, the phage attachment site attP, and to one of the two substrates for excisive recombination, the prophage-left site attL. Int binds to the phage attachment site with an esti- mated dissociation constant of lo-” M.

Bacteriophage X is a temperate virus with two pathways for its propagation, lytic or lysogenic. In the lysogenic pathway, the viral DNA is inserted into and replicated as a part of the host chromosome. The insertion process, termed integration or integrative recombination, is shown diagrammatically in Fig. 1 (1-4). Integration requires the product of the phage int gene, the Int protein. Integration occurs only at specific se- quences on X and host DNA called attachment sites. As shown in Fig. 1, the phage attachment site a.a’ (attP) and the host attachment site b-b‘ (attB) recombine to insert the viral genome. This creates two rearranged att sites: the prophage 6 . a’ (attL) near the galactose genes, and the prophage a - b’ (attR) near the biotin genes. For excision of X DNA, the

* This research was supported in part by Research Grant GM 17078 and Training Grant GM 07232 from the National Institute of General Medical Sciences, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Harvard University, Cambridge, Mass. 02138. $ Present address, The Biological Laboratories, 16 Divinity Ave.,

Gdansk, 24 Kladki, 80-822 Gdansk, Poland. 5 Present address, Department of Microbiology, University of

of Technology, Cambridge, Mass. 02139. 7 Present address, Department of Biology, Massachusetts Institute

prophage attachment sites recombine to regenerate the free phage DNA and the unoccupied host attachment site. Exci- sion requires the phage Int and Xis proteins. The DNA sequences of the att sites have been determined (5, 6). The central . in each attachment site represents a 15-base pair sequence that is a “common core” to each of the sites; each “arm” of the att sites, a,a’,b,b’, has a different sequence of nucleotides.

Other bacteriophage insert their genome into Escherichia coli, but at different sites on the host chromosome. These phages specify their own integration proteins, which must recognize different viral DNA sequences and different host sequences. Because of these sequence specificities, viral inte- gration is termed site-specifk recombination (1-4). Host genes have been found to affect the integration of X (7-9). However, the “host factors” defined by these genes are not specific for X integration; they affect integration by $80 and other tem- perate phage. Thus, in site-specific recombination there are elements of specific interaction, such as the X Int protein and the X attP DNA sequence, and elements which are nonspecific, such as the host factors.

The crucial specificity role for X Int protein has been dem- onstrated in vitro by the observation that Int protein binds much more tightly to DNA carrying the X attP site than to DNA deleted for the attP site or to DNA containing the att site of +BO (10, 11). The requirement for Int in site-specific recombination in vitro has been demonstrated by Nash and co-workers (11-13), as has the requirement for host proteins from uninfected cells.

In a previous paper (lo), we demonstrated that X Int protein could be detected and partially purified as a specific DNA- binding protein, using a binding assay dependent on retention of :’*P-labeled X DNA on a nitrocellulose fiter. We report here the use of this assay to purify X Int protein nearly to homo- geneity. Int purified as a DNA-binding protein catalyzes the complete integrative recombination reaction if host factors are added. We also describe some of the physical properties and binding characteristics of the purified Int protein. A purification of Int protein based on integrative recombination has been developed independently by Kikuchi and Nash (11).

EXPERIMENTAL PROCEDURES

Materials

Bacteriophage and Bacteria-The E. coli strain used was C6OOSu-

cIts857 (15), Sam7 (16), and intc226 (17). The intc mutation causes (suppressor-negative) (14). The phage point mutations used were

the constitutive production of the Int protein (in the absence of positive regulation by the X cII/cIII proteins) through activation of a promoter site (18). For the production of Int protein from wild type phage, CCiOOSu- cells were infected with XcI857 Sam7 (see “Results”). For the production of Int protein from intc phage, a lysogen of CGOOSu- (XcI857intc226Sam7) was subjected to thermal induction as

2433

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2434 Int Protein of Phage h

.II> X

gal b:b' bio -

gof 6.0' 0.b' bio

FIG. 1. Integration of h DNA into the host chromosome by site-specific recombination. The viral attachment site a . a' recom- bines with the bacterial attachment site b. b' to insert X DNA between the host galactose and biotin operons. For insertion, the products of the viral int gene and host genes are required. For the reverse reaction (excision), the products of the viral int and xis genes are required for recombination between the prophage attachment sites b.a' and a . b.

described under "Results." The phage deletion and/or substitution mutations used were: b538, deleting the phage attachment site (19); gaZ8, carrying the bacterial gal operon and the left prophage attach- ment site attL (20); bio7-20, carrying the bacterial bio operon and the right prophage attachment site attR (21); andgall49biol1, carry- ing both the gal and bio genes and the bacterial attachment site attB (22). The attachment site of each phage was verified by testing for the ability to transduce the appropriate galactose and/or biotin genes (see Fig. 1).

Proteins-Pancreatic DNase was obtained from Worthington and ovalbumin, chymotrypsin, myoglobin, and bovine serum albumin were from Schwarz/Mann.

Other Materials-Cellulose (CF11) and phosphocellulose (P11) were obtained from Whatman, and Sephadex G-75 (40 to 12-p particle size) were from Pharmacia. h DNA-cellulose was prepared as de- scribed by Alberts and Herrick (23). PMSF' was purchased from Calbiochem.

Methods Preparation of Phage [32P]DNA-"P-labeled phage DNAs were

prepared as described previously (24). In brief, cells were grown in 100 ml of low phosphate medium to a density of 5 X 10' cells/ml, concentrated by centrifugation, and infected with phage carrying the appropriate attachment site and the lysis-defective mutation Sam7 at a multiplicity of 5 phage/cell. After a 15-min adsorption period, the infected cells were poured into fresh low phosphate medium contain- ing 5 pCi/ml of "P-labeled inorganic phosphate. The cells were shaken at 37°C for 3 h and then collected by centrifugation and resuspended in 10 ml of 10 mM Tris-HCI (pH 7.2), 10 mM MgCL. The cells were lysed with chloroform, the debris was removed by centrif- ugation, and the supernatant fraction containing the phage was centrifuged to equilibrium in a CsCl density gradient. The phage band was removed with a syringe and the phage again centrifuged to equilibrium in CsCI. The phage DNA was extracted by four treat- ments with redistilled phenol, and the phenol was removed with ether. The DNA was then dialyzed extensively into 10 mM Tris-HC1

DNA-binding Assay-The DNA-binding assay for the Int protein was carried out as described previously (10). In brief, the assay measures the retention of "P-labeled DNA to nitrocellulose filters

of unlabeled "chicken blood" DNA (Calbiochem), which competes (B-6, Schleicher and Schuell). The assay mixture contains an excess

with the labeled phage DNA for proteins with nonspecific DNA- binding activity. Each assay contains 0.4 pg of phage ["*P]DNA, 20 pg of chicken blood DNA, and is 10 mM Tris-HC1 (pH 7.2), 70 mM KCl, 10 m~ MgCI2, 0.2 mM dithiothreitol, 0.2 mM EDTA in a total reaction volume of 100 pl. The various DNA preparations used gave 5 to 15% binding without any added extract; the binding data were corrected for this background. To distinguish fractions containing Int protein, parallel assays were done with attP DNA and DNA deleted

(pH 7.2), 0.1 mM EDTA.

' The abbreviations used are: PMSF, phenylmethylsulfonyl fluo- ride; SDS, sodium dodecyl sulfate.

for the att site. For the purification of Int protein the 50-fold excess chicken blood DNA was always used. In all of the experiments reported here using the highly purified enzyme, the chicken blood DNA was omitted; binding by the purified Int protein was specific even when the competitor DNA was removed.

Complete Integrative Recombination Reaction-Supercoiled plas- mid DNA carrying the phage attachment site (attP) will recombine with linear plasmid DNA carrying the bacterial attachment site (attB) in the presence of Int protein and host proteins from uninfected cells (25). Reaction mixtures (20 p1) contained 20 mM KCI, 5 mM spermi- dine, 5 rn EDTA, 50 mM Tris-HCI (pH 7.4), 0.25 pg each of attP and attB DNA, purified host factors (500 ng) (gift of H. Nash)? and Int protein ( 5 0 ng). After incubation at 25°C for 1 h, the reactions were stopped by the addition of SDS (final concentration, 0.5%), and agarose gel electrophoresis was carried out as described by Kikuchi and Nash (25). The assays were carried out with T. Pollack and C. Robertson, in the laboratory of H. Nash.

Miscellaneous Methods-Polyacrylamide gel electrophoresis of SDS-denatured protein was carried out in 15% polyacrylamide gels as described by Laemmli and Favre (26).

RESULTS

Purification of Int Protein

A summary of the purification of Int protein is presented in Table I. Unless noted otherwise, all operations were carried out at 0-4°C.

Growth of Infected or Induced Cells-For infection, a 100- liter culture of E. coli strain CGooSu- was grown in a New Brunswick fermenter at 37°C in 1% Difco tryptone, 0.5% yeast extract, 0.5% NaC1, 0.2% maltose, and 10 m MgC12. The p H was carefully kept between 7.0 and 7.2. When the A5w reached 0.7, the cells were infected with XcIts857Sam7 at a multipIicity of 10 to 15. After 20 min, the cells were harvested by centrif- ugation in a Sharples continuous flow centrifuge. The cell paste was frozen quickly in blocks of dry ice. One hundred liters generally yielded about 150 g of cells. For the production of induced lysogens, C600Su- cells lysogenic for intc226cIts857Sam7 were grown at 32°C to an ASw of 0.7. Phage development was initiated by raising the temperature t o 42°C for 15 min (denaturing the temperature-sensitive CI repressor). The temperature was then lowered t o 37°C for continued growth for 60 min.

Cell Lysis-Frozen cells (200 g) were cut up and blended into 40 ml of 1.0 M Tris-HC1 (pH 7.2), 80 mi of 2.0 M KCl, and 160 ml of distilled water. Before blending the protease inhib- itor, PMSF was added to a final concentration of 50 pg/ml. Lysozyme and EDTA were added to final concentrations of 0.15 mg/ml and 0.05 mM, respectively, and the cells were lysed by incubation for 30 min at 0°C. DNase and MgCL were added to final concentrations of 10 pg/ml and 10 mM, respec- tively, and the lysate was incubated at 32°C until the viscosity markedly decreased. The lysate was chilled to O"C, brought to 1.0 M KC1, and centrifuged for 16 h in a Spinco type 19 rotor at 17,000 rpm. The 1000-ml supernatant was dialyzed for 2 h against two changes of 5 liters of 10 mM KPOs (pH 6.4), 0.2 mM EDTA, 0.2 mM dithiothreitol, and 10% glycerol (Buffer KP). When the conductivity (measured with a Radi- ometer conductivity meter type CDM2e) reached that of Buffer K P containing 0.4 M KCl, the dialysis was stopped (crude extract Fraction I).

Phosphocellulose Chromatography-Fraction I was ap- plied to a phosphocellulose column (3.5 X 25 cm) previously equilibrated with KP buffer with 0.4 M KCI. The column was washed extensively with 700 ml or more of KP buffer with 0.4 M KC1 until the Azm of the effluent was less than 0.1. The column was eluted with a 600-ml gradient of 0.4 to 1.75 M KC1 in Buffer KP. The AZm and conductivities of the fractions

T. Pollack and H. Nash, manuscript in preparation.

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Int Protein of Phage X 2435

were taken, and assays were carried out for binding to X DNA carrying the substrate att site and to X DNA deleted for the att site. The DNA-binding activity specific for the X att site elutes between 0.8 and 1.0 M KC1 (Fraction 11) (Fig. 2). We have found that the sue of the column markedly changes the effectiveness of this purification step. The optimal situation is a 1:1 ratio of cells lysed (g) to column bed volume (ml). If the column is larger, the Int DNA-binding activity is not easily detected, presumably masked by other DNA-binding proteins; if it is smaller, the activity is not retained by the column.

Sephadex G- 75 Chromatography-Fraction I1 was dialyzed against Buffer KP with 0.4 M KCI. From this purification stage onward, the recovery of Int after dialysis can be quite variable; we have found that rapid dialysis against low salt (<0.1 M KCl) exacerbates this loss. The dialyzed Fraction I1 was concentrated by adsorption to a phosphocellulose column (1.0 X 3 cm) equilibrated with KP buffer with 0.4 M KC1 and eluted with KP buffer containing 1.3 M KC1 (concentration by ammonium sulfate precipitation gives a very poor recovery of DNA-binding activity and so was not used). This concentrated Fraction I1 was applied to a Sephadex G-75 column (2 X 110 cm) equilibrated with KP buffer with 0.2 M KC1. The flow rate of the column was 25 ml/h. The specific DNA-binding activity eluted slightly ahead of the position found for ovalbumin (M,

TABLE I Purification of Znt protein from intc phage

Fraction Volume Units" pro- Spe- cific Yield

tein activitv

ml mg units/mg I. Crude extract 800 - 13,500 -

11. Phosphocellulose 157 1,560 142 11 100 h -b

111. Sephadex 85 1,480 20 74 95 IV. DNA-cellulose 10.5 1,088 1.5 724 72

One unit is defined as the amount of Int protein required to retain 1 pg of X DNA to a filter at 0°C (to measure specific binding, the binding to att-deletion DNA is subtracted).

Due to the large number of DNA-binding proteins from E . coli, no atP-specific DNA binding can be detected at this step. - ,"I 1.2 .i-

h 60 -

0 0 - - c t 4)

4 0 - 4) a d - t 3 0 n a n

20 - z

i - E 0.8 E

0.4 - 0

0.8 Y

0 C

0

0.6 h

a

9"

- 0.4 o

Q

I ,\lo*2 0 to 20 30 40 5 0

Fraction number

0

FIG. 2. Phosphocellulose chromatography of Int protein. An extract from 200 g of induced lysogens (carrying an intc prophage) was fractionated and assayed for DNA-binding activity as described xnder "Experimental Procedures." w, binding to DNA contain- ing the viral attachment site (attP); 0---0, binding to DNA deleted fo; the viral attP site; A-A, Am; -, pooled fractions for Fraction I1 of Table I.

= 45,000) (Fraction 111) (Fig. 3). DNA Cellulose Chromatography-Fraction I11 was directly

applied to a A DNA-cellulose column (2 X 5 cm, containing 3 mg of DNA) equilibrated with KP buffer with 0.2 M KC1. The column was eluted with an 80-ml gradient of 0.15 to 1.0 M KC1 in KP buffer. The specific DNA-binding activity eluted at approximately 0.4 M KC1 (Fig. 4). The active fractions were pooled (Fraction IV), distributed into lOO-pl aliquots, and

V 0 i i i

M

50 60 70 80

0.3

0.2 5 0 (D

0.1 9"

Fraction number FIG. 3. Sephadex G-75 chromatography of Int protein. Frac-

tions from the phosphocellulose column of Fig. 2 containing DNA- binding activity specific for the phage attachment site were pooled, concentrated, chromatographed, and assayed for DNA-binding activ- ity. V, void volume; 0, elution position of ovalbumin (M, = 45,000); M, myoglobin (M, = 16,000). M, binding to DNA containing the viral attP site; O"-O, binding to DNA deleted for the viral attP site; A-A, AZm; I, pooled fractions for Fraction 111 of Table I.

" 10 15 20 25 30 35 " Fraction number

FIG. 4. DNA-cellulose chromatography of Int protein. The Sephadex fractions of Fig. 3 containing DNA-binding activity specific for attP were pooled and chromatographed on a DNA-cellulose column containing X DNA. +"O, binding to DNA containing the viral attP site; 0--0, binding to DNA deleted for the viral attP site; A-A, A,; t"~, pooled fractions for Fraction IV of Table I. When similar Sephadex fractions were chromatographed on DNA-cellulose containing salmon sperm DNA, virtually identical elution of A280 and binding activity were found (data not shown).

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2436 Int Protein of Phage h

frozen. If kept a t -20°C in either 50% or 10% glycerol (frozen), this fraction loses activity with a half-life of about 1 month. We have not stored Fraction IV long enough a t -70°C to assay its decay. Fraction IV was used in all the experiments presented in this paper.

Purity of the Final Product-When Fraction IV was ana- lyzed by electrophoresis in SDS on an acrylamide gel, one major band was found (Fig. 5). From electrophoresis of radio- active X proteins, several laboratories have reported independ- ent estimates for the molecular weight of Int protein of between 36,000 and 42,000 (27-31). Thus, previous evidence is consistent with the idea that this major band of M , = 40,000 is, in fact, the integration protein.

In purifying the Int protein from wild type and intc226 phage, we have not detected any difference in the chromato- graphic properties of the two proteins. After electrophoresis in SDS gels, we found no apparent difference in their sizes (Fig. 5). We have also found no difference in binding specificity to different attachment sites for integrative recombination (see below). Purification from intc phage is easier because of higher levels of Int protein and the absence of regulatory requirements, but we were concerned that the regulatory mutation might conceivably alter the int gene product. Al- though our results do not prove that the products from these two sources are identical, we think it likely because we have found no difference in any of the properties that we have examined.

Activity of Int Protein in the Complete Recombination Reaction-We wanted to know that the purification of Int as

C 1 0 0

FIG. 5. Tracings of SDS-polyacrylamide gels of purified Int protein (Fraction IV) f rom intc phage (top) and wild type phage (bottom). Approximately 5 pg of protein was used. Slab gel electrophoresis followed the methods of Laemmli a r ' Favre (26). After electrophoresis in a 15% SDS-polyacrylamide ge., the gel was stained with Coomassie brilliant blue and destained, and the protein hands were scanned with a Joyce-Loebl densitometer. Within the accuracy of the gel analysis, the Int protein from the two sources appears indistinguishable. The fractionation of molecular weight markers is indicated by W ~ O U J S : C, chymotr.ypsinogen ( M , = 25,000); 0, ovalbumin ( M , = 45,000); B, bovine serum albumin ( M , = 65.000).

a DNA-binding protein produced a product that was active in the complete recombination reaction. In collaboration with T. Pollock, C . Robertson, and H. Nash, we tested the purified Int in three different assays that require the complete integra- tion reaction in order to see the products. The data we present here are from the assay that we consider least subject to artifacts; it measures recombination between two plasmid DNA molecules, one containing the phage attachment site (attP) and the other carrying the bacterial attachment site (attB) (25). If integrative recombination occurs, the two plas- mid DNAs are joined to form the larger dimer DNA molecule that has a much slower mobility in electrophoresis on an agarose gel. Fig. 6 shows the result of such a reaction. Int purified as a DNA-binding protein catalyzes the complete reaction (Fig. 6b). Int protein purified by the complete inte- gration assay of Kikuchi and Nash (11, 25) generates the identical product (Fig. 6c). Neither Int alone (data not shown) nor host factors alone (Fig. 6a) can catalyze this reaction. In a second recombination-electrophoresis assay with both at- tachment sites on the same molecule (12), Int catalyzed intra-

Recombinant-

aft B

aft P L

Host factor

Int protein

+ + + -

+ +

FIG. 6. Assay for complete integration reaction. Supercoiled plasmid DNA containing the phage attachment site (attP) was incu- bated with linear DNA containing the bacterial attachment site ( n / t R ) and with Int protein and/or host factors. Integrative recombination generates the product of length equal to the sum of the substrates (Recombinant). The faint band below Recombinan/ is derived from nicked circles present in the original atlI' plasmid preparation. Chan- nel b used the Fraction IV Int protein of Table I. Channel c used Int protein provided by H. Nash that was prepared using the integrative recombination assay (1 1).

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Int Protein of Phage h 2437

molecular recombination (data not shown). Int was also active in a third recombination assay involving filter detection (13). Thus, Int purified as a DNA-binding protein can catalyze integrative recombination if supplemented with host factors from uninfected cells. We conclude that Int is the only phage- specified protein required for integrative recombination.

Sedimentation Properties of Int Protein-To estimate the molecular weight of native Int protein, we carried out velocity sedimentation in a 5 to 20% sucrose gradient (Fig. 7). The estimated sedimentation coefflcient is 2.6, as judged by the sedimentation of marker proteins of known S value. If Int has an axial ratio in the typical range for a globular protein, this sedimentation coefficient would indicate a molecular weight of 30,000 to 35,000, less than that calculated for the monomer in the SDS gel (see Fig. 5). Apparently, Int sediments as a monomer. The relatively low sedimentation rate even for a monomer might result from one or more of several sources: 1) the presence of a lower molecular weight form of Int protein, possibly a result of proteolysis; 2) an asymmetric shape for native Int; 3) a partial denaturation (unfolding) of Int during sedimentation. We cannot at present distinguish clearly among these possibilities. During Sephadex fractionation, most of the specific DNA-binding activity elutes slightly ahead of ovalbumin ( M , = 45,000); this result is consistent with an asymmetric shape, which should produce more rapid elution in gel fitration but slower sedimentation than ex- pected for a molecular weight of 40,000. However, Kikuchi and Nash (11) have reported a higher sedimentation coeffi- cient of 3.0. Evidently, complexities remain to be resolved in the sedimentation behavior of Int protein.

Binding Properties of Int Protein Attachment Site Specificity-There are four substrate (at-

tachment) sites that participate in site-specific recombination: the phage attP, the bacterial attB, the prophage-left attL, and the prophage-right attR (Fig. 2). We have analyzed the bind- ing of the purified Int protein to each of these sites (Fig. 8). The results demonstrate binding specificity for attP and attL; there is not a clear indication of binding specificity for attR or attB, because the binding curves for these potential substrates are not markedly different from that of the attachment site deletion (attA). Thus, we conclude that purified Int protein

0 m 0 5 10 15 20

Fraction number

FIG. 7. Velocity sedimentation of purified Int protein in a sucrose gradient. Int protein (0.2 ml of Fraction IV) was layered on a linear 5 to 20% sucrose gradient containing 0.6 M KC1, 0.1 mM EDTA, 0.2 mM dithiothreitol, and 50 mM Tris-HC1 (pH 7.2). Sedi- mentation was for 24 h in a SW 50.1 rotor at 49,000 rpm. Arrows indicate the sedimentation position of marker proteins in a separate gradient: B, bovine serum albumin (M, = 65,000); 0, ovalbumin (M, = 45,000); C, chymotrypsinogen (M, = 27,000), and M, myoglobin (Mr = 17,000). W, binding to DNA containing the viral attP site; M, binding to DNA deleted for the attP site.

80

60 c c 0) 0

0) P

L

$ 40

n a

3 0

z 0

20

n

r / /

v- 0 .05 .l 0 .15 .20

I n t concentration, pq/ml

FIG. 8. Binding of purified Int protein to different attach- ment sites. Int protein (Fraction IV from wild type phage) was added to labeled DNA (4 pg/ml) containing: the viral attP site (W); the bacterial attB site (M); the prophage-left attL site

for the attP site (attA DNA) (M). (A-A); the prophage-right attR site (A-A); and DNA deleted

-i c"

I I I I I 2 4 6 8 10

DNA concentration, pq/ml

FIG. 9. Equilibrium DNA-binding curve for Int protein (Fraction IV from intc phage) with DNA containing: the viral attP site (M); and DNA deleted for the attP site (M). The concentration of Int was held constant (approximately 1 pg/ml) and the DNA concentration was varied.

recognizes one of the two substrates in integrative recombi- nation (attP) and in excisive recombination (attL). Similar results and conclusions have been derived by Kikuchi and Nash (11).

From earlier experiments with partially purified Int, we noted specific binding to attP and attL, but we also found apparent binding to attB (10). We believe that the apparent attB binding was not a property of Int protein.

Estimation of Equilibrium Dissociation Constant-The data of Fig. 8 suggest that the binding specificity of Int protein for attP and attL is sufficient to estimate an equilibrium dissociation constant. This is most conveniently done from a

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2438 Int Protein of Phage X

binding curve in which DNA concentration is varied (32-34). Data are given for Int protein from intc phage (Fig. 9); an identical binding curve is found for Int protein from wild type phage (data not shown). If we assume that there is one high affinity binding site per DNA molecule, we calculate a disso- ciation constant of 2 X lo-“ M. Although comparisons with dissociation constants for other DNA-binding proteins are difficult to interpret because of variations in ionic strengths, buffers, and temperature, the binding of Int appears to resem- ble the “weak repressor” h Cro protein (34) more closely than the “strong repressors,” XcI protein (35) and Lac repressor (32), which have dissociation constants of about M.

DISCUSSION

The Int protein of phage h is of considerable interest for an understanding of viral growth and its regulation because it is the specificity determinant for the localized genetic recombi- nation event that generates the prophage state. Because other systems of localized genetic recombination exist and because the initiation events are not understood for any type of genetic recombination, a knowledge of the way in which Int protein works is also likely to provide important insight into mecha- nisms for other types of genetic exchange. As a fist step toward this understanding, we have developed a convenient and reproducible purification for Int protein and initiated a study of the specific interaction of this protein with DNA.

We have shown that purified Int binds specifically to the phage attP and prophage-left attL attachment sites (Fig. 1). Thus, Int determines recombination site, at least in part, by binding specificity for the substrate sites, presumably defining the initiation point for the reaction. However, two major problems remain; how do the other substrate sites, attB and attR, enter the reaction, and how is the breaking and joining accomplished?

With regard to substrate sites, another phage protein, Xis, is required for the excision reaction in which attL and uttR recombine; Xis might provide recognition specificity for attR. However, no evidence exists for another phage protein re- quired for the integration reaction, and purified Int is the only phage-specified protein required for the complete integrative recombination reaction in vitro (this work and Ref. 11). Thus, the recognition of attB poses a problem. Possibilities include the following: 1) a host protein binds to attB (and then to Int); 2 ) a host protein interacts with Int to allow Int to recognize attB; 3) weak binding to uttB, not detectable by the filter assay (36), is sufficient; 4) a special conformation of Int or substrate is required for effective attB recognition. We favor the fourth possibility because of the high specificity of site- specific recombination for both phage and host sites and because it seems unlikely that such host specificity involves a number of different host proteins (one for each temperate phage). Two examples of such “conformation mechanisms” are: Int binds first to attP and itself undergoes a conformation change, allowing tight binding to attB; or Int acts in the right side of attP to open the helix at the common core, allowing effective recognition of the common core of attB. The detailed interaction of Int with attP is clearly complex (36, 37); the mechanism for pairwise substrate recognition will undoubt- edly require detailed studies at the nucleotide level.

We cannot provide any experimental insight so far into the nature of the breaking and joining. Because of the requirement for superhelicity for integrative recombination in vitro (12, 13), a plausible model is one in which the “recognition” protein(s) brings the DNA strands together and a “nicking- closing” activity analogous to L.J protein or gyrase consum- mates the reaction (nicking “opposite” strands instead of the already demonstrated nicking of “same” strands). Kikuchi

and Nash (25) have recently shown that purified Int protein preparations have topoisomerase (nicking-closing) activity and have provided evidence that this activity involves Int protein itself. We have also found topoisomerase activity in our purified Int preparation. Thus, Int protein may carry out both the recognition and breaking-joining aspects of the re- action (25). Alternatively, Int protein may utilize the topoiso- merase activity to generate the appropriate substrate for the breaking and joining reaction (e.g. an unwound common core able to base pair (38)).

Acknowledgments-We thank Howard Nash for the gift of strains and the communication of results prior to publication, and Howard Nash, Thomas Pollock, and Carol Robertson for their hospitality to M. K. at the National Institutes of Health and assistance with the assays for the complete reaction for integrative recombination.

REFERENCES 1. Gottesman, M. E., and Weisberg, R. A. (1971) in The Bacterio-

phage Lambda (Hershey, A. D., ed), pp. 113-138, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

2. Echols, H. (1971) Annu. Rev. Biochem. 40,827-854 3. Nash, H. (1978) Curr. Top. Microbiol. Immunol. 78, 17-199 4. Weisberg, R. A., Gottesman, S., and Gottesman, M. E. (1977) in

H., eds) Vol. 8, pp. 197-258, Plenum Press, New York Comprehensive Virology (Ftaenkel-Conrat, H., and Wagner,

5. Landy, A., and Ross, W. (1977) Science 197, 1147-1160 6. Davies, R. W., Schreier, P. H., and Buchel, D. E. (1977) Nature

7. Miller, H. I., and Friedman, D. I. (1977) in DNA Insertion Elements, Plasmids, and Episomes (Shapiro, J., Bukhari, A., and Adhya, S., eds), pp. 349-356, Cold Spring Harbor Labora-

8. Williams, J. G. K., Wulff, D. L., and Nash, H. A. (1977) in DNA tory, Cold Spring Harbor, New York

Insertion Elements, Plasmids, and Episomes, (Shapiro, J., Bukhari, A,, and Adhya, S., eds), pp. 357-361, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

9. Miller, H., Kikuchi, A,, Nash, H., Weisberg, R., and Friedman, D. (1978) Cold Spring Harbor Symp. Quant. Biol. 43, 1121-1127

10. Kotewicz, M., Chung, S., Takeda, Y., and Echols, H. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 1511-1515

11. Kikuchi, Y., and Nash, H. A. (1978) J. Biol. Chem. 253, 7149- 7157

12. Mizuuchi, K., and Nash, H. A. (1976) Proc. Natl. Acad. Sci. U. S.

13. Nash, H. A., Mizuuchi, K., Weisberg, R., Kikuchi, Y., and Gellert, M. (1977) in DNA Insertion Elements, Plasmids, and Epi- somes (Shapiro, J., Bukhari, A., and Adhya, S., e&) pp. 363- 373, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

14. Court, D., Green, L., and Echols, H. (1975) Virology 63, 484-491 15. Sussman. R.. and Jacob, F. (1962) C. R. Hebd. Seances Acad. Sci.

270, 757-760

A. 73,3524-3528

254, 1517-1519 16. Goldbere. A. R.. and Howe. M. (1969) Virolom 38,200-202 17. Shimada,’K., ani! Campbell, A. (1974) Viroli& 60, 157-165 18. Fischer, R., Takeda, Y., and Echols, H. (1979) J . Mol. Biol. 129,

19. Parkinson, J. S., and Huskey, R. J. (1971) J. Mol. Biol. 56, 369-

20. Feiss, M., Adhya, S., and Court, D. L. (1972) Genetics 71,189-206 21. Manly, K. F., Signer, E. R., and Radding, C. M. (1969) Virology

22. Nash, H. A. (1974) Virology 57,207-216 23. Alberts, B., and Herrick, G. (1971) Methods Enzymol. 21, 198-

24. Wu, A. M., Ghosh, S., EchoIs, H., and Spiegelman, W. G. (1972)

25. Kikuchi, Y., and Nash, H. A. (1979) Proc. Natl. Acad. Sci. U. S.

26. Laemmli, U. K., and Favre, M. (1973) J. Mol. Biol. 80, 575-599 27. Nash, H. A. (1974) Nature 247,543-545 28. Ausubel, F. M. (1974) Nature 247, 152-154 29. Hendrix, R. (1971) in The Bacteriophage Lambda (Hershey, A.

D., ed), pp. 355-370, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

30. Court, D., Adhya, S., Nash, H., and Enquist, L. (1977) in DNA

509-514

384

37, 177-188

217

J. Mol. Biol. 67,407-421

A. 76,3760-3764

by guest on February 17, 2018http://w

ww

.jbc.org/D

ownloaded from

Int Protein of Phage X 2439

Znsertion Elements, Plasmids, and Episomes (Shapiro, J., Bukhari, A., and Adhya, S., eds), pp. 389-394, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

31. Katzir, H., Oppenheim, A., Belfort, M., and Oppenheim, A. B. (1976) Virology 74,324-331

32. Riggs, A. D., Suzuki, H., and Bourgeois, S. (1970) J. Mol. Biol.

33. Wilcox, G., Clemetson, K. J., Cleary, P., and Englesberg, E. (1974)

34. Takeda, Y., Folkmanis, A., and Echols, H. (1977) J. Biol. Chem.

48,67-83

J. Mol. Biol. 85,589-602

252,6177-6183 35. Chadwick, P., Pirrotta, V., Steinberg, R. A., Hopkins, N., and

Ptashne, M. (1970) Cold Spring Harbor Symp. Quant. Bwl. 35,

36. Ross, W., Landy, A,, Kikuchi, Y., and Nash, H. (1979) Cell 18,

37. Davies, R. W., Schreier, P. H., Kotewicz, M. L., and Echols, H.

38. Champoux, J. J. (1977) Proc. Natl. Acad. Sei. U. S. A. 74, 5328-

283-294

297-307

(1979) Nucleic Acids Res. 7, 2255-2273

5332

by guest on February 17, 2018http://w

ww

.jbc.org/D

ownloaded from

M Kotewicz, E Grzesiuk, W Courschesne, R Fischer and H Echolsbacteriophage lambda.

Purification and characterization of the integration protein specified by

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