Suicide Recombination Substrates Yield Covalent X Integrase-DNA

THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 263, No. 16, Issue of June 5, pp. 7678-7685,1988 Printed in U.S.A.

Suicide Recombination Substrates Yield Covalent X Integrase-DNA Complexes and Lead to Identification of the Active Site Tyrosine*

(Received for publication, November 9, 1987)

Christopher A. Pargellis, Simone E. Nunes-Diiby, Lina Moitoso de Vargas, and Arthur LandyS From the Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912

High levels of covalent integra*-DNA complexes accumulate when suicide substrates containing a medial nick within the overlap region are nicked by A integrase protein. The tyrosine residue at position 342 is shown to form a covalent bond with DNA at the sites of strand exchange. A mutant integrase in which this tyrosine is changed to phenylalanine is devoid of both topoisomerase and recombinase activity but still binds to both core- and arm-type DNA binding sites with an affinity comparable to wild-type integrase. Tyrosine- 342 is located within a 40-amino acid region that is conserved among 15 known recombinases comprising the “integrase family.” The present results show that this small region of homology participates in catalysis of strand transfer.

The study of reaction intermediates is often complicated by virtue of their transient and unstable nature. In the present work we apply the rationale of mechanism based inactivators (Helmkamp et al., 1968) to the well characterized site-specific recombination system of bacteriophage lambda. Treatment of pre-nicked DNA suicide substrates with X Int’ leads to the production of reaction intermediates in the form of covalently bound protein-DNA complexes.

Lambda integrative recombination between specific sites on the phage (attP) and bacterial (attB) chromosomes re- quires the phage encoded 40,000-dalton Int protein and the bacterially encoded integration host factor (IHF) (for reviews see Nash, 1981; Weisberg and Landy, 1983). The sequential exchange of top and bottom strands occurs at sites that are separated by 7 base pairs (Mizuuchi et al., 1980b; Nunes- Duby et al., 1987). The nucleotide sequence between the two sites can be varied, but recombining partners must retain identical sequences (Weisberg et al., 1983; Bauer et al., 1985). This 7-bp “overlap” region flanked by two inverted “core- type” Int binding sites constitutes the 25-bp G B site (ROSS and Landy, 1983; Mizuuchi and Mizuuchi, 1985). The more complex 240-bp &tP contains several additional protein binding sites for IHF and Int (“arm-type” Int sites) (Craig and Nash, 1984; Ross and Landy, 1982).

Purified Int protein nicks att site DNA at the sites of strand exchange (Craig and Nash, 1983), resolves synthetic att site

* This work was supported by National Institutes of Health Grant GM33928. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed. The abbreviations used are: Int, integrase; IHF, integration host

factor; attP, phage attachment site; attB, bacterial attachment site; TEMED, N,N,N’,N’-tetramethylethylenediamine; SDS, sodium do- decyl sulfate; bp, base pair(s).

Holliday junctions (Hsu and Landy, 1984), and acts as a type I topoisomerase (Kikuchi and Nash, 1979). The absence of a cofactor requirement implied that the free energy associated with phosphodiester bond hydrolysis is preserved in aprotein- DNA intermediate for subsequent use during ligation (Wang, 1971). Although a covalent link between Int and the 3’-side of its specific nick site has been demonstrated (Craig and Nash, 1983), the very low levels of this complex (less than 1% of input DNA) made further analysis difficult.

This r2port describes a family of suicide recombination substrates that are pre-nicked within the overlap region. Int forms a covalent bond when it specifically nicks these substrates; however, the reaction is unable to proceed and covalent Int-DNA complexes accumulate. Using a combination of biochemical and genetic analyses, it is shown that tyrosine- 342 is the residue forming this covalent bond with DNA. These results are discussed in the context of other recombinases that share a region of amino acid homology which we propose to be part of the active site.

EXPERIMENTAL PROCEDURES

Plasmids and Proteins-Plasmid DNA preparations and transfor- mations were carried out as described previously (Ross and Landy, 1983). Restriction digestions and ligations were performed as recom- mended by the supplier (New England Biolabs). Int protein was produced from expression plasmids under the control of the tac promoter in Escherichia coli strain K5298 ( h i d A 81 ladQ Tn9 gal-) constructed by Ellen Woodland’ (Rhode Island Hospital) from strains K5242 and K5598-W3110 that were obtained from Harvey Miller (Genentech, San Francisco). Cells grown in LB broth to an OD,, of 0.3 to 0.4 were induced with 1 mM isopropyl-0-D-thiogalactopy- ranoside (Behring Diagnostics) and grown for an additional 3-4 h before harvesting. Int and IHF were purified to homogeneity (>98%) by modifications of published procedures (Kikuchi and Nash, 1978; Nash and Robertson, 1981) that will be described elsewhere. Protein concentrations were determined by the dye-binding method (Brad- ford, 1976).

Preparation of 3ZP-Labeled DNA Substrates with a Medial Nick- The design and construction of functional att site analogs with an XbaI restriction site within the overlap region, and flanking XhoI and EcoRI sites to the left and right, respectively, have been described (Nunes-Diiby et at., 1987). In pSN109 attB the XbaI-staggered nicks are located at the left strand exchange site (top strand) and at the center of the overlap region (bottom strand). As diagrammed in Fig. 1, this plasmid was cleaved with XhoI and then resected by the 3’- exonuclease activity of Tq DNA polymerase (OFarrell et al., 1980) in the presence of 0.1 mM dATP for 5 min at 37 “C, followed by addition of EDTA to 10 mM, and heat inactivation of the enzyme. The DNA was recovered by ethanol precipitation before a second round of controlled resection by T, DNA polymerase in the presence of 0.1 mM dCTP. After a third round of resection in the presence of [a-”P] dGTP (Du Pont-New England Nuclear) for 5 min, an excess of 4 unlabeled dNTPs was added and incubation was continued for 60 min to resynthesize the 18 nucleotides of the bottom strand of attB. Restriction with XbaI and PstI yielded a 760-bp right-half attB fragment singly labeled at the right exchange site (bottom strand).

* Ellen Woodland, unpublished results.

7678

Identification of the Active Site Tyrosine in X Int 7679

This fragment was gel-purified and mixed with a 5- and 10-fold excess of gel-purified left-half attB (320-bp BamHI-XbaI) and full length attB (1080-bp BamHI-PstI) fragments, respectively. The DNA mix- ture was denatured in 0.2 M NaOH, 10 mM EDTA, and reannealed as described (Nunes-Diiby et al., 1987). Two new nicked 1080-bp products are generated, only one of which is labeled with 32P (see Fig. 1). An equivalent labeling and annealing scheme, but starting with the pSN108 attB analog, is used to prepare the suicide substrate with a top strand medial nick and 32P label at the top strand exchange site (not illustrated).

P,-nuclease Digestion and Identification of Phosphoamino Acids- Nicked substrates incubated with Int (25-50 nM) under recombination conditions (Nunes-Duby et al., 1987) for 1 to 2 h yielded approximately 50% Int-DNA complexes. These were digested with PI- nuclease (Pharmacia LKB Biotechnology Inc.) a t 200 units/ml after adjusting the buffer conditions to 30 mM ammonium acetate (pH 5.3), 0.2 mM ZnClz at 60 "C for 1 h. Some heat precipitation of proteins (Int and/or bovine serum albumin) was apparent. The reaction was stopped by the addition of EDTA to 10 mM. Int-DNA complexes were recovered by continued heat precipitation at 65 to 70 "C for 1 h followed by centrifugation. Analysis for 3ZP-amino-acids was performed by modifications of known procedures (Shriner and Brautigan, 1984). 32P-Int protein was treated with 6 N HCI at 110 "C for 2 h in an evacuated sealed tube. The hydrolysate was applied onto a 20 X 20 X 0.3-mm cellulose thin layer plate (Kodak) in presence of Thr(P), Ser(P), and Tyr(P) (10 pg each). Electrophoresis a t 900 V (45 V/cm) was carried out on a Pharmacia flatbed apparatus at pH 1.9 for 2 h and in the second dimension at pH 3.5 for 75 min (Hunter and Sefton, 1980).

Formic Acid Cleauage of 3ZP-Znt-The pellet collected after centrifugation of heat-precipitated 3ZP-labeled Int protein was resuspended in 100 p l of 70% formic acid, which is known to favor cleavage between Asp-Pro residues (Landon, 1977). 0.8 mg (20 nmol) of unlabeled Int protein was added before incubation at 37 'C for 36 h. Despite the inability to completely solubilize Int protein by this procedure, very little intact protein remained after the 36-h incubation. Similar observations have been reported for related peptide cleavages under acidic conditions (Inglis, 1983; Bergmeyer et al., 1981). Formic acid-cleaved peptides were dried by vacuum centrifugation and dissolved in 100 p1 of 6% acetic acid using sonication. This was applied to a 1.0 X 48-cm Fractogel TSK HW-50(F) column pre- equilibrated in 6% acetic acid and run at a flow rate of 2 ml/h. Those fractions containing the desired 32P-peptide were pooled and chro- matographed a second time.

SDS-Urea Polyacrylamide Gel Electrophoresis-Int protein and related large molecular weight peptides were analyzed by standard discontinuous SDS-polyacrylamide gel electrophoresis using 12.5% gels (Ames, 1974). Low molecular weight peptides were resolved on SDS-urea polyacrylamide gels (Swank and Munkries, 1971), using the cyanogen bromide cleavage products of apomyoglobin and cyto- chrome c as molecular weight markers. 20-cm X 20-cm X 1-mm continuous gels contained 12.5% acrylamide, 0.83% N,N'-methylene- bis-acrylamide, 8 M urea, 0.1% SDS, 0.1 M Tris phosphate (pH 6.8), 0.052% ammonium persulfate, and 0.005% TEMED. Samples were concentrated by vacuum centrifugation and were dissolved in 10 pl of a loading solution containing 8 M urea, 10 mM Tris phosphate (pH 6.8), 1% 2-mercaptoethanol, 1% SDS, and bromphenol blue. Samples were denatured by heating at 70 "C for 10 min prior to electrophoresis at 200 V for 14 h.

NHz-terminal Peptide Sequencing-Sequencing of the peptides was carried out by the Sequence Facility at the University of California, Davis, on a Beckman Model 890 M liquid phase sequencer. Amino acids were identified as their phenylthiohydantoin derivatives using two different reverse-phase high performance liquid chromatography systems.

Construction of pLV342F and pLV318F-The Int mutant Y342F, in which Tyr-342 is replaced by phenylalanine, was constructed with a 92-bp (double-stranded) synthetic oligomer composed of 9 overlap- ping single-stranded oligonucleotides and containing an A to T substitution in codon 342. For purposes of screening, a new EcoRI site was also introduced into the synthetic oligomer. The oligomer was inserted into pCL280, an int expression vector under the control of the tac promoter: by replacing the 113-bp fragment extending from the NcoI site at codons 337/338 to a Hind111 site just after the int coding region. The resulting plasmid is pLV342F. The Int mutant Y318F, in which Tyr-318 is replaced by phenylalanine, was con-

Cammie Lesser, unpublished results.

structed with an 85-bp double-stranded synthetic oligomer containing an A to T substitution in codon 318 and a new BglII site for purposes of screening. This was introduced into pLV356-1, an int expression vector under the control of the tac promoter4 by replacing the 85-bp fragment extending from the unique BamHI site at codons 303-304 to the AuaI site at codons 331/332. The resulting plasmid is pLV318F. Both constructions were initially transformed into W3110 lacIQ L8 (Brent and Ptashne, 1981) and subsequently into K5298.' The correct clones were identified by restriction digests and confirmed by sequencing the relevant regions (Maxam and Gilbert, 1980).

Assays of Int Function-Nuclease protection experiments were carried out as described previously (Thompson et al., 1987). Topoi- somerase activity was determined in a 20-pl assay containing 25 mM Tris-HC1 (pH 7.9), 5 mM EDTA, 2 mM dithiothreitol, 75 mM sodium chloride, 0.5 mg/ml bovine serum albumin and 0.3 pmol of supercoiled attP (pWR1) at an 1nt:att site molar ratio of about 301. Recombinase assays used the same buffer conditions with the addition of 6 mM spermidine and 1-2 units of IHF (20 to 40 nM or an 1nt:IHF molar ratio of about 2:l). 1 unit of IHF activity was defined, by titration with 1 unit of Int, as the smallest amount of protein which gives maximal recombination. The substrate DNAs, EcoRI-linearized attB (pWR101,5697 bp) and supercoiled attP (pWR1,4510 bp) (Bushman et al., 1984), were both at a concentration of 0.1 pmol per 20-111 assay. Reactions were incubated at 23-25 "C for 12-16 h at an 1nt:att site molar ratio of about 30:l and analyzed by electrophoresis on 1.2% agarose gels (Thompson et al., 1987).

RESULTS

Covalent Attachment of Int to Suicide Substrates-The suicide recombination substrates used in the present experiments have a pre-existing nick near the center of the overlap region. These substrates are prepared from recombination proficient uttB analogs containing an XbuI site within the left (pSNlO9) or right (pSNlO8) boundary of the overlap region (Nunes-Duby et al., 1987). As diagrammed in Fig. 1, a pSNlO9 uttB fragment is subjected to resection and resynthesis in order to insert a single 32P label at the bottom strand phosphodiester linkage normally attacked by Int (right exchange site) (see "Experimental Procedures"). In the annealing scheme used to prepare suicide substrates, a large excess of unlabeled intact att DNA ensured that most of the radioactive strands are incorporated into the medially nicked suicide substrate (as opposed to reforming half-utt fragments; see Fig. 1 and Fig. 2, lane I).

Incubation of medially nicked suicide substrates from pSNlO9 with Int protein produces an increase in the amount of protein-free right-half att site and a new band of lower electrophoretic mobility (Fig. 2, lune 2 ) . SDS-KC1 precipitation of the sample (Trask et al., 1984) yields a proteinaceous pellet enriched in the slower band (lane 3 ) and a supernatant containing two species of protein-free DNA (lane 4 ) . Incuba- tion of the suicide substrate with a top strand medial nick (derived from pSN108) with Int gives rise to a similar band of lower electrophoretic mobility (lanes 5 and 7) precipitable by SDS-KC1 (data not shown). This slower moving band is removed by phenol extraction which is apparent as a loss of total radioactivity (lune 6). Pronase digestion also abolishes the slower moving band, but with a transfer of radioactivity to a band that migrates the same as unreacted substrate (lune 8). These findings suggest that Int is covalently linked to att site DNA, causing a mobility shift during electrophoresis. The efficiency of the suicide substrates in forming aborted covalent Int-DNA complexes is highlighted when they are used in a normal integration reaction with Int and IHF. Int-DNA Complexes are the major product; less than 15% of the input radioactivity appears in nicked circular recombination intermediates or linear recombinant^.^

' Lina Moitoso de Vargas, unpublished results. ' Simone Nunes-Duby, unpublished results.

7680

X b a I /

u n n i c k e d - h a l f a r m s e

e x c h a n g e s i t e n i c k

lnt. P-

Identification of the Active Site Tyrosine in X Int

Xhol a, X b a I

L r e s e c t f i l l In

mix I X b a I 5 x d e n a t u r e -

s u b s t r a t e s su ic ide

1 L I I I I I I 1 1 I I I I I I I I I I 1 1 1 1 1 I I I I I I I I I I I I I I I I I I I I I I I I I *

kp-1 n t

u n 2 L 1 I

~ ~ I I I I I I I I I I I ~ ;I/ ?;:I:: I I I ' FIG. 1. Preparation of internally labeled suicide substrates

and suggested mechanisms for the formation of covalent Int- DNA complexes. attB DNA (pSNlO9) linearized with XhoI was resected across the overlap region (thick bars) and filled in with T, DNA-polymerase (wavy lines) using [a-"PIdGTP and unlabeled dNTPs (see "Experimental Procedures"). The half-att fragments produced by XbaI cutting were mixed with the original uncut attB in the proportions indicated (Zx, 5x, and ZOX), denatured, and reannealed so as to produce two types of nicked molecules. The suicide substrate (boxed) containing a medial overlap nick in the bottom strand and a single label (asterisk) at the site of strand exchange (curued open arrows) is incubated with Int. This leads to Int nicking the top strand and producing two fragments (kjt) or to Int nicking the bottom strand and generating a diffusible trimer (right).

Identification of a Tyr(P) Linkage-The covalent Int-DNA complex from the substrate nicked in the bottom strand and 3ZP-labeled at the bottom strand exchange site was digested extensively with P, nuclease to remove DNA and heat-precipitated at 65 "C. The resuspended radioactive pellet co-migrated with unlabeled Int protein on a 12.5% SDS-polyacrylamide gel, further indicating that "P was covalently linked to Int. "P-Labeled Int was hydrolyzed in 6 N HCl for 2 h at 110 "C. This hydrolytic procedure, shortened from the more typical 10- to 24-h cleavage, was found to maximize hydrolysis of both peptide and phosphonucleotide bonds, while minimiz- ing hydrolysis of the Tyr(P) bond. Analysis of the hydrolysate by two-dimensional thin layer electrophoresis in the presence of phosphoamino acid standards identified a major "'P-labeled spot which co-migrated with Tyr(P) (Fig. 3).

Mapping the Active Site Tyrosine-Inspection of the amino acid sequence of Int reveals 14 tyrosine residues as well as two acid-labile Asp-Pro sites. Cleavage between Asp-28 and Pro-29 generates a 3,200-dalton peptide from the amino terminus of Int containing 4 tyrosine residues. The second

"* " '4

T

SDS-KC1 C T P S

c-

*A- t

4 Phenol P r o n a s e - + - +

- Int-at t - - a t t - - - m h "half a t t

1 2 3 4 5 6 7 0

FIG. 2. Gel electrophoresis of "P-labeled suicide substrates after incubation with Int. The substrates with a medial nick in the bottom strand (pSNlO9; lejt) or top strand (pSN108; right) are diagrammed above each panel. The annealing scheme used to prepare these prenicked att sites was identical to that described in Fig. 1, but the fragments were end-labeled using polynucleotide kinase (asterisks). The unreacted pSNlO9 sample ( l o n e c ) contains mostly suicide substrate (att) carrying radioactivity acquired from the XbaI-cleaved fragments (half-att) during annealing. (The small, labeled half-att fragment of pSNlO8 has run off the gel.) After a 2-h incubation with Int ([am T), Int-DNA complexes (Znt-att) are recovered in the pellet (P) with SDS-KCI, leaving protein-free DNA in the supernatant (S) . Aliquots of the equivalent Int-pSN108 reaction were electrophoresed before (-) and after (+) phenol extraction or Pronase digestion.

I

-m. P-Ser

....... -" tT P-Thr N.. . -

RG. 3. Phosphoamino acid analysis of '*P-labeled Int. '*P- Int obtained by PI-nuclease digestion of labeled Int-DNA complexes was subjected to acid hydrolysis and two-dimensional electrophoresis as described under "Experimental Procedures." The positions of the standards (Ser(P) (P-Ser), Thr(P) (P-Thr), Tyr(P) (P-Tyr); dotted circles) were determined by staining with ninhydrin and the "P located by autoradiography. The origin is indicated with a circle at bottom right. Several fainter spots not corresponding to the unlabeled standards probably result from the incomplete hydrolysis of peptide bonds (Gronostajski and Sadowski, 1985) and/or phosphonucleoside bonds.

cleavage between Asp-303 and Pro-304 results in a central 30,600-dalton peptide containing 8 tyrosine residues and a 6,400-dalton carboxyl-terminal peptide with 2 tyrosines (Fig. 4).

The "P-labeled covalent Int-DNA complex was digested with PI-nuclease, heat-precipitated, mixed with excess unla-

7681

+ 303’304 Aap-PrO-Pro-Thr-Pha-~ia-QlU-Lau-Arg-Sar-LaU--8ar--A1a-Arg-Leu-Tyr-Qlu-Ly~-Qln-I1a-Bar-A~p-

308 311 3 18

+ + + + + + + + #Is - - 446 X/S THU - 4 1 4 - - LEU - - - C I Y - - - * * 1 3 7 3 93 8 7 8 7

+ Ly~-Pha-Al~-Qln-lli~-Lau-Uu-Qly-lli~-Lya-8ar-Aap-Thr-Wat-Al.”Oln-Tgr-Qln-~r-------------L

342 356 Y.

- - I C E C I X - - ibu CIY #/I - - - - - - - - ryu 8 7 87 8 0 100 8 0 *

FIG. 4. Amino acid sequence of lambda integrase showing the conserved carboxyl-terminal portion from the integrase family of recombinases. The primary sequence of Int protein (356 amino acids) was determined from the DNA sequence of the int gene (Hoess et al., 1980). After cleavage with 70% formic acid at the two Asp-Pro sites (arrowheads), both the NHz-terminal and COOH-terminal peptides were identified by NH,- terminal sequencing (underlining arrows). The two Tyr residues in the COOH-terminal peptide (diamonds) were individually altered to phenylalanine by site-directed mutagenesis to generate the Int mutants Y318F and Y342F. The conserved region for the 15 known members of the integrase family has been aligned with the Int sequence. Three positions are identical in all 15 cases (asterisks). The remaining residues were analyzed by placing them in one of six exchange groups: Ser, Pro, Ala, Gly, Thr; Arg, Lys, His; Phe, Trp, Tyr; Asp, Glu, Gln, Asn; Ile, Leu, Met, Val; and Cys (Dayhoff et al., 1978). When at least 73% (11 out of 15) of the residues at a given location were in the same exchange group (percentage shown in the bottom line), the predominant conserved amino acid is shown in italics. Levels of similarity of 50 to 70% are present in 12 of the other positions (hyphens).

beled Int protein, and incubated with formic acid (see “EX- perimental Procedures”). When this hydrolysate was fractionated by size-exclusion chromatography, a total of 81% of the applied radioactivity was recovered. The first large peak (Fig. 5, peak I) contains only 29% of the recovered radioactivity. This fraction is expected to contain uncleaved Int, three large peptides resulting from cleavage at either or both of the two Asp-Pro sites, and large peptides due to cleavage at bonds of intermediate acid lability. Examination of the autoradiograph does not reveal any major radioactive species.

The second peak from the size-exclusion column contains 71% of the recovered radioactivity. SDS-urea polyacrylamide gel electrophoresis of this fraction shows a single silver- stained peptide of 5 to 6 kDa and a major band of radioactivity that migrates slightly slower (Fig. 5, middle and bottom). This mobility difference is presumably due to retardation of the labeled peptide by the covalently linked nucleotide that is not present in the unlabeled carrier Int. Similar observations of retarded mobilities of 32P-peptides relative to unlabeled carrier peptides have been reported elsewhere (Horowitz and Wang, 1987; Sugino et al., 1980).

The fractions in peak I1 were pooled and rechromato- graphed on the same column. The resulting single peak was homogeneous by SDS-urea polyacrylamide gel electrophoresis with respect to both radioactivity and carrier protein. The NH2-terminal sequence of the first 10 residues indicates that this peptide starts at Pro-304. Its molecular weight is consistent with a peptide extending to the carboxyl terminus of Int as predicted (cf. Fig. 4).

The third major peak eluting from the column had no 32P and was shown by SDS-urea polyacrylamide gel electrophoresis to contain a single peptide with an apparent molecular mass of 2.5 to 3.0 kDa (Fig. 5). The sequence of the first 10 residues of this peptide matches the NH, terminus of intact Int with the NH2-terminal methionine removed and its molecular mass is consistent with a formic acid catalyzed cleavage between Asp-28 and Pro-29 (cf. Fig. 4).

These results limited the number of possible active site tyrosines to those two located in the carboxyl-terminal formic acid peptide. Further biochemical approaches to the resolu-

tion of Tyr-318 and Tyr-342 were hampered by the fact that, as the peptides are reduced in size, the chromatographic and electrophoretic differences between 32P-labeled and unlabeled peptides compromise the validity of final peptide identification. For this reason the two tyrosine candidates were resolved by genetic analysis.

Substitution of Tyr-318 and Tyr-342 with Phenylalanine- Site-directed mutagenesis was used to construct two mutant int genes, containing phenylalanine in place of either Tyr-318 (Y318F) or Tyr-342 (Y342F) (see “Experimental Proce- dures”). Cell extracts containing wild-type, Y318F, or Y342F Int were assayed for topoisomerase and recombinase activity (Fig. 6). Y318F has retained both topoisomerase and recombinase activity while Y342F has completely lost both of these activities. To rule out the possibility of specific inhibitory effects of the extract, the Y342F protein was purified to homogeneity and shown to be still defective. The purified Y342F was also studied in nuclease protection experiments and shown to bind to both arm-type and core-type Int binding sites with affinities comparable to those of wild-type Int (Fig. 7). Thus, it is Tyr-342 and not Tyr-318 that forms the covalent bond with DNA.

DISCUSSION

The results reported here, which allow the identification of a key residue in the catalytic site of a recombinase, are based upon the use of suicide recombination substrates containing a medial nick in the overlap region. Several observations indicate their relevance to the normal recombination path- way. First, it has been shown that XbaI containing att analogs (with and without an XbaI nick in attB) are functionally equivalent to the canonical att sites (Nunes-Duby et al., 1987). Second, by labeling a single phosphate in the overlap region (Fig. l), it has been shown that the covalent linkage is at the same phosphodiester bond that is cut during the strand exchange of recombination (Mizuuchi et al., 1980b) and during the nicking of a canonical att site (Craig and Nash, 1983). Finally, as discussed below, the active site Tyr-342 identified in these experiments is consistent with conserved protein sequence patterns found in 14 other recombinases (Argos et

7682 Identification of the Active Site Tyrosine in X Int

HCOOH Cleaved Intograre TSK HW-(IO(F) (1 M CHJCOOH)

17.200 b

14,700 b

7,760 b

6,420 b

2,780 b 2.550 b

40,000 b

17,200 b

14,700 b

7,710 b

1.420 b

2.710 b

FIG. 5. Purification of 32P-labeled peptide from the Int-DNA complex treated with 70% HCOOH. Upperpanel, 0.8 mg of the RYP-labeled Int-DNA complex that had been hydrolyzed in 70% HCOOH was fractionated by chromatography on a size-exclusion gel in 6% CHnCOOH as described under "Experimental Procedures." The absorbance a t 280 mM (thin line) and the radioactivity (thick discontinuow lim) are indicated for relevant fractions (number located on the abscissa). Middle and lower panels, samples were loaded onto 12.5% polyacrylamide gels in the presence of 0.1% SDS and 8 M urea. The gels were fixed and silver-stained (middle panel) prior to autoradiography (lower panel). The gel has been overstained to better visualize the low molecular weight peptides.


wt INT Y318F Y342F

FIG. 6. Topoisomerase and recombinase assays. Topoisom- erase assays, with supercoiled attP ( P s.c.) (bottom panel), and recombinase assays, with both supercoiled attP and linear attB ( B ) (top panel), were carried out on the indicated cell extracts as described under “Experimental Procedures.” Equal volumes of extract (arrows) used in both types of assays are indicated in microliters. Products of each assay are shown as recombinant (rec.) or relaxed attP ( P rel.). All three extracts contained approximately the same concentration of protein: wt Int = 15.5 mg/ml; Y318F = 14.5 mg/ml; and Y342F = 14.0 mg/ml.

al., 1986) and with genetic analyses on 2 of them (see below). Previously, covalent Int-DNA complexes were extremely

difficult to isolate. Presumably they are short lived and rapidly give rise to ligated product. This is consistent with the broad distribution of topoisomers generated by Int, suggesting that only a small number of supercoils are released before religa- tion (Kikuchi and Nash, 1979; Mizuuchi et al., 1980a). High yields of covalent Int-DNA complexes are obtained with medially nicked suicide substrates. It is likely that nicking by Int at the site of strand exchange either cuts prenicked attB in half or generates a 3-base oligomer that is not expected to remain hydrogen-bonded at 25 “C (see Fig. 1). Diffusion of the half-site, or the trimer, deprives Int of the 5’-hydroxyl it would normally use as an acceptor in the ensuing ligation reaction, thus trapping the covalent intermediate.

A common motif of proteins that have the capacity to cut and reseal DNA is conservation of phosphodiester bond energy in a transient covalent protein-DNA linkage. In each of the six topoisomerases analyzed at this level, the DNA is linked to the protein through a Tyr(P) (Tse et al., 1980; Champoux, 1981; Rowe et al., 1984, Brougham et al., 1986; Horowitz and Wang, 1987) and for E. coli DNA gyrase the active tyrosine has recently been mapped (Horowitz and Wang, 1987). Proteins involved in replication priming mechanisms of several unrelated phages (“terminal proteins”) have been shown to form covalent protein-DNA linkages via any of the three hydroxy amino acids, tyrosine (Bamford and Mindich, 1984), serine (Desiderio and Kelly, 1981; Hermoso et al., 1985) and threonine (Garcia et al., 1986). In the 4x174 gene A protein two tyrosines separated by three amino acids have been shown to form covalent bonds to DNA in an apparently alternating pattern, during the cleavage and ligation reactions required for initiation and termination of roll- ing circle replication (van Mansfeld et al., 1986). The site- specific recombinases appear to fall into at least two families, based on the chemistry of strand transfer. One of these is

P I P2

Core

P‘ 1

P’ 2

P’3

urn a

”

“ c

FIG. 7. Nuclease protection with wild-type Int and the Y342F mutant. Nuclease protection experiments were carried out with homogeneous Int and using neocarzinostatin to digest the DNA as described previously (Thompson et al., 1987). The “bottom strand” of the NcoI to Hind111 fragment of pWRl was labeled with 3zP at the 5’-end. The Int protein binding sites are indicated (left-hand margin) (Ross and Landy, 1982, 1983). Dilutions of Int used in each reaction are shown at the top of each lane with the undiluted reactions (labeled I ) containing 22.4 fmol of wild-type Int and 17.3 fmol of Y342F mutant Int. The central lane (-) contains no protein.

represented by y6 Resolvase (Grindley et al., 1984), where genetic studies have implicated serine-10 (Hatfull and Grind- ley, 1986) as a likely candidate for the known Ser(P) linkage with DNA (Reed and Moser, 1984). Lambda Int belongs to another family called the “integrase family” (Argos et al., 1986).

The results presented here show that Tyr-342 of lambda Int forms a covalent linkage with the 3”phosphate at the site(s) of strand exchange. Substitution of Tyr-342 with phenylalanine produces a mutant Int that completely lacks both recombinase and topoisomerase activity. However, Y342F binds to both arm-type and core-type sites with affinities comparable to those of wild-type Int, suggesting that there is little or no perturbation of tertiary structure in this mutant.

The integrase family of recombinases is defined by small regions of homology located near their carboxyl termini (Ar- gos et al., 1986). In addition to the 8 recombinases originally used to define the integrase family (Argos et al., 1986), seven new proteins have been identified. The presently known members include the integrases of temperate bacteriophages lambda (Hoess et al., 1980), 480, P22 (Leong et al., 1986), 186

7684 Identification of the Active Site Tyrosine in X Int

(Kalionis et al., 1986), P2,6 P4 (Pierson and Kahn, 1987), HKOB7; Cre of P1 (Sternberg et al., 1986); the putative Tn2603/R46 recombinase (Ouellette and Roy, 1987; Hall and Vockler, 1987); a pair of invertases controlling fimbrial phase variation in E. coli, FimB and FimE (Klemm, 1986; Eisenstein et al., 1987); the F-factor D-protein, ResD' (O'Connor et al., 1986; sequenced by R. Eichenlaubg); tnpA and tnpB of the Staphylococcus aureus transposon Tn5547 (Murphy et al., 1985) and the 2-pm plasmid FLP-protein from Saccharomyces (Hartley and Donelson, 1980). Recently, five additional plasmids from another yeast species, Zygosaccharomyces, have been isolated which code for recombinases closely related to FLP (Utatsu et al., 1987).

Within this family of 15 proteins one region of approximately 40 amino acids contains three perfectly conserved residues, His-308, Arg-311, and Tyr-342. At 22 additional positions within this region, more than 50% of the residues belong to the same amino acid exchange group (Dayhoff et al., 1978). A mutational analysis of one of the members of this family, Cre protein from bacteriophage P1, showed that single substitutions at any one of the three perfectly conserved positions destroy in vitro recombination activity but do not affect binding (Wierzbicki et al., 1987). In another member of this family, FLP protein of the Saccharomyces cereuisiae 2- pm circle, amino acid substitutions for the proposed active site tyrosine also abolished nicking activity without diminish- ing DNA binding (Prasad et al., 1987). FLP has been shown to form a Tyr(P) bond with DNA, although the specific tyrosine involved has not been mapped (Gronostajski and Sadowski, 1985).

All of the 15 recombinases catalyze closely related reactions although the mechanistic requirements and DNA binding functions differ widely. The positions of sequence homology shared among these proteins may be correlated with catalytic function, while the largely divergent sequences may be part of other domains with different roles. Indeed, we have recently separated two peptide domains of X Int that possess unique properties consistent with this The identification of Tyr-342 as the active nucleophile in Int lends strong support to the proposal that the conserved carboxyl-terminal region is primarily involved in the actual catalysis of the nicking and resealing reactions. This result constitutes biochemical evidence that correlates catalytic function with the conserved carboxyl-terminal region in the integrase family of recombinases.

Acknowledgments-We wish to thank Robert Weisberg, Michael Malamy, David Friedman, and R. Eichenlaub for communicating unpublished results. We are also grateful to David Brautigan and Robert Weisberg for helpful discussions, Carol Egner for the purification of Y342F, Joan Boyles, Bonnie Tracy, and Lucy Rodrigues for technical assistance.

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Suicide Recombination Substrates Yield Covalent X Integrase-DNA