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Site-specific Photo-cross-linking between λ Integrase and Its DNA Recombination Target

2002; Elsevier BV; Volume: 277; Issue: 17 Linguagem: Inglês

10.1074/jbc.m108197200

1083-351X

Margaret J. Kovach, Radhakrishna S. Tirumalai, Arthur Landy,

Advanced biosensing and bioanalysis techniques

The site-specific recombinase (Int) of bacteriophage λ is a heterobivalent DNA-binding protein and is composed of three domains as follows: an amino-terminal domain that binds with high affinity to “arm-type” sequences within the recombination target DNA (att sites), a carboxyl-terminal domain that contains all of the catalytic functions, and a central domain that contributes significantly to DNA binding at the “core-type” sequences where DNA cleavage and ligation are executed. We constructed a family of core-type DNA oligonucleotides, each of which contained the photoreactive analog 4-thiodeoxythymidine (4-thioT) at a different position. When tested for their respective abilities to promote covalent cross-links with Int after irradiation with UV light at 366 nm, one oligonucleotide stood out dramatically. The 4-thioT substitution on the DNA strand opposite the site of Int cleavage led to photo-induced cross-linking efficiencies of ∼20%. The efficiency and specificity of Int binding and cleavage at this 4-thioT-substituted core site was shown to be largely uncompromised, and its ability to participate in a full site-specific recombination reaction was reduced only slightly. Identification of the photo-cross-linked residue as Lys-141 in the central domain provides, along with other results, several insights about the nature of core-type DNA recognition by the bivalent recombinases of the λ Int family. The site-specific recombinase (Int) of bacteriophage λ is a heterobivalent DNA-binding protein and is composed of three domains as follows: an amino-terminal domain that binds with high affinity to “arm-type” sequences within the recombination target DNA (att sites), a carboxyl-terminal domain that contains all of the catalytic functions, and a central domain that contributes significantly to DNA binding at the “core-type” sequences where DNA cleavage and ligation are executed. We constructed a family of core-type DNA oligonucleotides, each of which contained the photoreactive analog 4-thiodeoxythymidine (4-thioT) at a different position. When tested for their respective abilities to promote covalent cross-links with Int after irradiation with UV light at 366 nm, one oligonucleotide stood out dramatically. The 4-thioT substitution on the DNA strand opposite the site of Int cleavage led to photo-induced cross-linking efficiencies of ∼20%. The efficiency and specificity of Int binding and cleavage at this 4-thioT-substituted core site was shown to be largely uncompromised, and its ability to participate in a full site-specific recombination reaction was reduced only slightly. Identification of the photo-cross-linked residue as Lys-141 in the central domain provides, along with other results, several insights about the nature of core-type DNA recognition by the bivalent recombinases of the λ Int family. The integrase (Int) 1The abbreviations used are: Intintegrase4-thioT4-thiodeoxythymidinentnucleotideHPLChigh pressure liquid chromatographyCBcore bindingMOPS4-morpholinepropanesulfonic acid1The abbreviations used are: Intintegrase4-thioT4-thiodeoxythymidinentnucleotideHPLChigh pressure liquid chromatographyCBcore bindingMOPS4-morpholinepropanesulfonic acid protein of bacteriophage λ, which was first purified by Kikuchi and Nash (1.Kikuchi Y. Nash H.A. J. Biol. Chem. 1978; 253: 7149-7157Abstract Full Text PDF PubMed Google Scholar), belongs to a large family of site-specific recombinases (the λ Int family) that rearrange DNA sequences having little or no sequence homology. λ Int is a heterobivalent site-specific DNA-binding protein and a type I topoisomerase that catalyzes the insertion and excision of the viral genome into and out of the hostEscherichia coli genome (for reviews see Refs. 2.Nash H.A. Escherichia coli and Salmonella.in: Neidhardt F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella, Cellular and Molecular Biology. American Society for Microbiology, Washington, D. C.1996: 2363-2376Google Scholar, 3.Hallet B. Sherratt D.J. FEMS Microbiol. Rev. 1997; 21: 157-178Crossref PubMed Google Scholar, 4.Grainge I. Jayaram M. Mol. Microbiol. 1999; 33: 449-456Crossref PubMed Scopus (119) Google Scholar). Integrative recombination between specific target sites on the phage (attP) and bacterial (attB) chromosomes generates an integrated prophage bounded by attL and attR sites at the junctions of bacterial and phage DNA. Excisive recombination between attL and attR recreates theattP and attB sites and yields free viral DNA (5.Campbell A.M. Caspari E.W. Thoday J.M. Advances in Genetics. Academic Press, New York1962: 101-145Google Scholar) (see Fig. 1). integrase 4-thiodeoxythymidine nucleotide high pressure liquid chromatography core binding 4-morpholinepropanesulfonic acid integrase 4-thiodeoxythymidine nucleotide high pressure liquid chromatography core binding 4-morpholinepropanesulfonic acid Each att site contains an inverted pair of “core-type” Int-binding sites (9 bp each) with a centered 7-bp “overlap region.” A reciprocal exchange of the “top” strands at the left boundary of the overlap region generates a Holliday junction recombination intermediate, which is then resolved by exchange of the “bottom” strands at the right boundary of the overlap region. DNA cleavage is mediated by a tyrosine hydroxyl that attacks the scissile phosphate, forming a 3′-phosphotyrosine link to the nicked DNA. This covalent protein-DNA intermediate is resolved when the 5′-terminal hydroxyl of the invading DNA strand attacks the phosphotyrosine linkage and displaces the protein. The chemistry of DNA strand exchange and the general arrangement of the “core region” are common to all of the λ Int family members except that their overlap regions vary from 6 to 8 bp in length, and their core-type binding sites vary from 9 to 13 bp. For some family members, such as Cre, XerC/D, and FLP, the core region composes the entire (minimal) att site. For other family members, such as λ and HP1 integrases, the att sites are more complex and contain additional protein-binding sites in viral DNA sequences that compose flanking “arms.” Some of these flanking sites bind to DNA bending accessory proteins like IHF, Xis, and Fis, whereas others bind to the amino-terminal domain of Int, resulting in a higher order complex where Int bridges the core and arm sequences of a sharply bent att DNA. Underlying the various layers of complexity that divide λ Int family members into several subgroups is a common carboxyl-terminal catalytic domain that executes the cleavage and rejoining of core-type DNA sequences in an energy-conserving reaction pathway that is the hallmark of these recombinases. The carboxyl-terminal “domain” of λ Int (residues 65–356) binds with low affinity to core-type sites located at the positions of strand cleavage and functions as a topoisomerase. This C65 can be further dissected by proteolysis into two smaller domains, encompassing residues 65–169 and 170–356, respectively. The latter, termed C170 or the catalytic domain, has been characterized and its crystal structure has been determined (6.Tirumalai R.S. Healey E. Landy A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6104-6109Crossref PubMed Scopus (43) Google Scholar, 7.Kwon H.J. Tirumalai R.S. Landy A. Ellenberger T. Science. 1997; 276: 126-131Crossref PubMed Scopus (178) Google Scholar). The C170 domain contains all of the residues that have been identified as being conserved in the λ Int family of recombinases (8.Esposito D. Scocca J.J. Nucleic Acids Res. 1997; 25: 3605-3614Crossref PubMed Scopus (260) Google Scholar, 9.Nunes-Düby S. Tirumalai R.S. Kwon H.J. Ellenberger T. Landy A. Nucleic Acids Res. 1998; 26: 391-406Crossref PubMed Scopus (365) Google Scholar, 10.Argos W. Landy A. Abremski K. Egan J.B. Haggård-Ljungquist E. Hoess R.H. Kahn M.L. Kalionis W. Narayana S.V.L. Pierson L.S.I. Sternberg N. Leong J.M. EMBO J. 1986; 5: 433-440Crossref PubMed Scopus (375) Google Scholar, 11.Blakely G.W. Sherratt D.J. Mol. Microbiol. 1996; 20: 234-237Crossref PubMed Scopus (16) Google Scholar). These include a very highly conserved triad of Arg-212, His-308, and Arg-311 that has been suggested to activate the scissile phosphate for DNA cleavage (12.Chen J.-W. Lee J. Jayaram M. Cell. 1992; 69: 647-658Abstract Full Text PDF PubMed Scopus (137) Google Scholar, 13.Pan G. Luetke K. Sadowski P.D. Mol. Cell. Biol. 1993; 13: 3167-3175Crossref PubMed Scopus (46) Google Scholar), the active site nucleophile Tyr-342 (14.Pargellis C.A. Nunes-Düby S.E. Moitoso de Vargas L. Landy A. J. Biol. Chem. 1988; 263: 7678-7685Abstract Full Text PDF PubMed Google Scholar), and Glu-174, which can be mutated to give a hyper-recombination phenotype (15.Lange-Gustafson B.J. Nash H.A. J. Biol. Chem. 1984; 259: 12724-12732Abstract Full Text PDF PubMed Google Scholar). The C170 domain of λ Int is approximately the same size as the smallest λ Int family members, such as FimB and FimE of E. coli (227 and 209 amino acids respectively) (16.Klemm P. EMBO J. 1986; 5: 1389-1393Crossref PubMed Scopus (264) Google Scholar). Crystal structures of the catalytic domain of HP1 integrase (17.Dyda F. Hickman A.B. Jenkins T.M. Engelman A. Craigie R. Davies D.R. Science. 1994; 266: 1981-1986Crossref PubMed Scopus (709) Google Scholar), the XerD recombinase (18.Subramanya H.S. Arciszewska L.K. Baker R.A. Bird L.E. Sherratt D.J. Wigley D.B. EMBO J. 1997; 16: 5178-5187Crossref PubMed Scopus (173) Google Scholar), and the Cre recombinase complexed with itsatt site loxA (19.Guo F. Gopaul D.N. Van Duyne G.D. Nature. 1997; 389: 40-46Crossref PubMed Scopus (476) Google Scholar) reveal protein folds resembling that of the λ Int C170 domain (7.Kwon H.J. Tirumalai R.S. Landy A. Ellenberger T. Science. 1997; 276: 126-131Crossref PubMed Scopus (178) Google Scholar). However, there are significant differences in the active sites of these recombinases, including different orientations of the tyrosine nucleophile and differences in nearby segments that are predicted to contact the DNA. In all of the λ Int family members there is a second domain just upstream of the catalytic domain that is also involved in binding to core-type sites. In λ Int this is called the central core binding (CB) domain because it is flanked by the carboxyl-terminal (catalytic) and amino-terminal (arm-binding) domains. In the monovalent λ Int family members that bind only to core-type sites (e.g. Cre, Flp, and XerC/D), the analogous domain is an amino-terminal domain. Previous studies in our laboratory (6.Tirumalai R.S. Healey E. Landy A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6104-6109Crossref PubMed Scopus (43) Google Scholar) had shown that although the catalytic domain of λ Int catalyzes the cleavage and rejoining of core-type DNA sequences, it does not form electrophoretically stable complexes with att DNAs. Experiments specifically designed to identify the interface(s) between λ Int and core-type DNA all pointed to the CB domain as follows: zero length UV-induced photo-cross-linking identified Ala-125 and Ala-126, DNA-sensitive modification by pyridoxal 5′-phosphate identified Lys-103, and an isolated CB domain formed stable complexes with core-type DNA (20.Tirumalai R.S. Kwon H. Cardente E. Ellenberger T. Landy A. J. Mol. Biol. 1998; 279: 513-527Crossref PubMed Scopus (45) Google Scholar). These results were unexpected because a number of experiments from other laboratories all pointed to the primacy of the catalytic domain in binding to core-type DNA. Additionally, several mutations affecting core-type DNA binding affinity and sequence recognition specificity were also found to be located in the catalytic domain (21.Dorgai L. Yagil E. Weisberg R.A. J. Mol. Biol. 1995; 252: 178-188Crossref PubMed Scopus (60) Google Scholar, 22.MacWilliams M.P. Gumport R.I. Gardner J.F. Genetics. 1996; 143: 1069-1079Crossref PubMed Google Scholar). The catalytic domains of Cre and FLP can bind autonomously to core-type sites, i.e. in the absence of their amino-terminal domains, which correspond to the CB domain of λ Int (23.Hoess R. Abremski K. Irwin S. Kendall M. Mack A. J. Mol. Biol. 1990; 216: 873-882Crossref PubMed Scopus (47) Google Scholar, 24.Panigrahi G.B. Sadowski P.D. J. Biol. Chem. 1994; 269: 10940-10945Abstract Full Text PDF PubMed Google Scholar). Finally, the Cre/loxA cocrystal structure indicated that upon binding tolox (core-type) DNA, the catalytic domain buries ∼50% more solvent-accessible surface area at the DNA interface than the amino-terminal (CB-analogous) domain (19.Guo F. Gopaul D.N. Van Duyne G.D. Nature. 1997; 389: 40-46Crossref PubMed Scopus (476) Google Scholar). To explore further these apparent dichotomies, we looked for a different chemistry with which to probe the Int-core DNA interface(s). We found that incorporation of the photoaffinity cross-linking analog, 4-thio-deoxythymidine (4-thioT), at a unique position in the core DNA yields high efficiency photo-cross-linking to Int. Identification of the photo-cross-linked residue as Lys-141 in the CB domain provides, along with other results, several insights about the nature of core-type DNA recognition by the bivalent recombinases of the λ Int family. An additional bonus of these experiments is the very high efficiency with which the photo-cross-link is generated, thus providing a potentially useful handle for dissecting the higher order organization of the large complex structures associated with this site-specific recombination pathway. Full-length bacteriophage λ Int, Int C65Y, and Int C65F proteins were produced inE. coli strain BL21 from expression plasmids under the control of bacteriophage T7 promoter and were purified from the insoluble (λ Int) and soluble (C65Y and C65F forms) fractions of the cell lysate (20.Tirumalai R.S. Kwon H. Cardente E. Ellenberger T. Landy A. J. Mol. Biol. 1998; 279: 513-527Crossref PubMed Scopus (45) Google Scholar). IHF (25.Nash H.A. Robertson C.A. J. Biol. Chem. 1981; 256: 9246-9253Abstract Full Text PDF PubMed Google Scholar, 26.Borowiec J.A. Zhang L. Sasse-Dwight S. Gralla J.D. J. Mol. Biol. 1987; 196: 101-111Crossref PubMed Scopus (178) Google Scholar) and Xis (27.Abremski K. Gottesman S. J. Biol. Chem. 1982; 257: 9658-9662Abstract Full Text PDF PubMed Google Scholar, 28.Yin S. Bushman W. Landy A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1040-1044Crossref PubMed Scopus (56) Google Scholar) were also purified from overproducing strains. The protein preparations, purified by a series of phosphocellulose (Whatman), SP-Sepharose (Amersham Biosciences), and hydroxylapatite (Calbiochem) column chromatography, were determined to be greater than 95% pure as evaluated by Coomassie staining of an overloaded SDS-polyacrylamide gel. Protein concentrations were estimated by the dye-binding method (29.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214351) Google Scholar). Synthetic oligonucleotides were purchased from Cruachem, Inc. (Dulles, VA), in lyophilized form. Before use, the oligonucleotides were resuspended in TE buffer at 1 mm. The sequences for each 4-thioT-substituted core-binding site (underlined) are as follows (where ts designates top strand, bs designates bottom strand, X = 4-thioT, and ↓ shows site of Int cleavage): T3 (ts, 23 nt), 5′-CTAXAAAGTTGCTCGAGAAGATC-3′; T3 (bs, 22 nt), 5′-GATCTTCTCGAGCAACTTT↓ATA-3′; T4 (ts, 34 nt), 5′-GTTCTAGXAAGTTGCTCGAGAAGATCTTCAGCTT-3′; T4 (bs, 30 nt), 5′-AAGCTGAAGATCTTCTCGAGCAACTTA↓CTA-3′; B4 (ts, 23 nt), 5′-CTATAAAGTTGCTCGAGAAGATC-3′; B4 (bs, 22 nt), 5′-GATCTTCTCGAGCAACTTX↓ATA-3′; B5 (ts, 23 nt), same as B4 (ts); B5 (bs, 22 nt), 5′-GATCTTCTCGAGCAACTXT↓ATA-3′; B6 (ts, 23 nt), same as B4 (ts); B6 (bs, 22 nt), 5′-GATCTTCTCGAGCAACXTT↓ATA-3′; T8 (ts, 34 nt), 5′-GTTCTAGAAAGXTGCTCGAGAAGATCTTCAGCTT-3′; T8 (bs, 30 nt), 5′-AAGCTGAAGATCTTCTCGAGCAACTTT↓CTA-3′; T9 (ts, 34 nt), 5′-GTTCTAGAAAGCXGCTCGAGAAGATCTTCAGCTT-3′; T9 (bs, 30 nt), 5′-AAGCTGAAGATCTTCTCGAGCAGCTTT↓CTA-3′; T11 (ts, 23 nt), 5′-CTATAAAGTTG XACGAGAAGATC-3′; T11 (bs, 22 nt), 5′-GATCTTCTCGTACAACTTT↓ATA-3′; B11 (ts, 23 nt), 5′-CTATAAAGTTGAACGAGAAGATC-3′; B11 (bs, 22 nt), 5′-GATCTTCTCGTX CAACTTT↓ATA-3′; T12 (ts, 23 nt), 5′-CTATAAAGTTGAXCGAGAAGATC-3′; T12 (bs, 22 nt), 5′-GATCTTCTCGATCAACTTT↓ATA-3′; B12 (ts, 23 nt), same as B11 (ts); B12 (bs, 22 nt), 5′-GATCTTCTCGXTCAACTTT↓ATA-3′; heterologous competitor (ts, 23 nt), 5′-CTAGAGAACGTCTCGAGAAGATC-3′; heterologous competitor (bs, 22 nt), 5′-GATCTTCTCGAGACGTTCTCTA-3′; non-TattL (27 nt), 5′-TCGAGCAGCTTT↓TTTATATTAAGTTGG-3′; 4ST-attL (27 nt), 5′-TCGAGCAGCTTT↓TTTATAXTAAGTTGG-3′; and PBR327 number 4 primer (20 nt), 5′-CTGCCACCA- TAGGCACGCCG-3′. Oligonucleotides used in binding and cross-linking assays were 5′-labeled using T4 polynucleotide kinase with [γ-32P]ATP. Complementary oligonucleotides were annealed by heating to 90 °C for 10 min in 10 mmTris·HCl (pH 7.5), 1 mm EDTA, 100 mm NaCl, followed by a slow cooling/annealing period of 4–16 h. Unincorporated [γ-32P]ATP was removed by passage through a Sephadex G-50–80 spin column, prior to gel purification of annealed DNA substrates (passive elution from a 10% polyacrylamide gel into TE buffer). Care was taken not to expose 4-thioT-substituted oligonucleotides to ultraviolet light. Radiolabeled half-att site DNA substrates (5 pmol) were incubated at 25 °C in a 10-μl mixture of 10 mm Tris·HCl (pH 7.5), 50 mm NaCl, 5% glycerol, 1 mm EDTA, 1 mg/ml bovine serum albumin, and 1 μg/ml sheared salmon sperm DNA. The reaction was initiated by the addition of Int C65Y (25 pmol) and allowed to proceed for 1 h. The reactions were stopped by the addition of 0.2% SDS buffer. Cleavage products were separated by electrophoresis through 10% polyacrylamide in 0.5× TBE, 0.1% SDS buffer. The gels were autoradiographed on Fuji x-ray film. Binding of Int C65F (25 pmol) to radiolabeled half-att site DNA substrates modified with 4-thioT substitutions proceeded at 25 °C for 20 min in a 10-μl mixture of 10 mm Tris·HCl (pH 7.5), 50 mm NaCl, 5% glycerol, 1 mm EDTA, 1 mg/ml bovine serum albumin, and 1 μg/ml sheared salmon sperm DNA. The samples were then transferred to the surface of a porcelain plate pre-chilled to 4 °C. Photo-cross-linking of the samples was achieved by exposing the samples to a 366 nm UV light source (Blak-Ray lamp, model UVL-56, Ultra-Violet products, Inc., San Gabriel, CA) positioned 2 cm away from the samples. Photo-cross-linking was performed on ice at 4 °C for a period of 45 min. Non-covalent interactions were disassociated by addition of 0.2% SDS. Photo-cross-linking of an Int C65F titration was also performed as described above, except that a constant amount of radiolabeled T3 duplex (5 pmol) was mixed with Int C65F (500, 250, 125, 60, 30, 15, and 7.5 pmol) prior to cross-linking. UV-dependent covalent complexes were separated by electrophoresis through 10% polyacrylamide gel in 0.5× TBE, 0.1% SDS buffer. The gels were exposed on a PhosphorImager plate and also autoradiographed on Fuji x-ray film. The PhosphorImager plate was scanned with a Fuji BAS 1000 scanner, and the image was visualized and quantitated with the Fuji MacBAS software package. Several concentrations of Int C65F (3, 6, 12, 25, and 50 pmol) were mixed with radiolabeled core-type oligomers (25 pmol) and incubated at 25 °C in a 10 μl mixture of 10 mmTris·HCl (pH 7.5), 50 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, and 5% glycerol. The reactions were analyzed by electrophoresis through an 8% polyacrylamide gel in 25 mm Tris, 200 mm glycine, 1 mm EDTA buffer. The gels were exposed on a PhosphorImager plate and also autoradiographed on Fuji x-ray film. The PhosphorImager plate was scanned with a Fuji BAS 1000 scanner, and the image was visualized and quantitated with the Fuji MacBAS software package. Competition binding assays were set up as described above, except that Int C65F (12.5 pmol) was incubated with radiolabeled T3 4-thioT duplex (25 pmol) in the presence of increasing amounts of non-labeled competitor DNA (25, 50, 75, 100, 125, and 150 pmol). The homologous competitor shared the same nucleotide sequence as the T3 4-thioT duplex except that it did not contain the 4-thioT substitution. The heterologous competitor shared the same base composition as the T3 duplex, but the sequence for the core binding domain was scrambled (see above). The gels were quantitated on the PhosphorImager. attL andattR plasmids containing the overlap sequences 5′-TTTATAX (pPEG1) and 5′-TTTATAT (pPEG2), respectively, were generated to replicate the overlap sequence of the T3 4-thioT half-att site. (Note that a bold X indicates the 4-thioT substitution.) 4-ThioT-substituted and non-substitutedattL substrates were generated by PCR amplification using the appropriate attL primer along with a downstream primer homologous to a sequence within the pBR327 backbone of the plasmid pPEG1 (see under “Oligonucleotides”). The attL substrates (∼400 bp) were gel-purified and 5′-labeled using T4 polynucleotide kinase with [γ-32P]ATP. Unincorporated [γ-32P]ATP was removed by passage through a Sephadex G-50–80 spin column. Recombination assays were carried out at 25 °C for 4 h in a 20-μl mixture containing 0.10 pmol of supercoiledattR DNA substrate and 0.05 pmol of radiolabeled linearattL substrate (4-thioT-modified or non-modified), 25 mm MOPS (pH 7.9), 50 mm NaCl, 5 mmEDTA, 6 mm spermidine, 2.5 mm dithiothreitol, and 0.5 μg/ml bovine serum albumin. Reactions were preincubated with 1.5 units of IHF for 20 min before the addition of 1.5 units of Xis and λ Int (8, 4, 2, and 1 pmol; 1.25 pmol λ Int is equivalent to 1 activity unit). Reactions were stopped with the addition of 0.2% SDS. Recombination products were analyzed by electrophoresis through 1.2% agarose gels that were then dried down and quantitated on the PhosphorImager. C65F (150 nmol) was photo-cross-linked to the T3 oligomer (125 nmol) in 1 ml of 10 mm Tris·HCl (pH 7.5), 50 mm NaCl, 1 mm EDTA, and 5% glycerol for 45 min at 4 °C after binding for 20 min at 25 °C. The sample was concentrated down to approximately a 200-μl volume (Centricon 30, Amicon, Inc.) and washed 5 times with 2-ml volumes of 8 m urea, 100 mmNH4HCO3 by Centricon dialysis. This serves to denature the protein as well as to remove a significant amount of the non-cross-linked DNA. The sample was then diluted to 2 murea by the addition of 3 volumes of 100 mmNH4HCO3, to make the conditions compatible with trypsin digestion. The digestion was allowed to go to completion (∼24 h), and the Int C65F photo-cross-linked to T3 4-thioT-attDNA was resolved by anion-exchange HPLC on a Sychrom AX 300 (250 × 4.6 mm) column, equilibrated with 20 mm sodium phosphate (pH 6.8), 20% (v/v) acetonitrile (equilibration buffer). The peptides were eluted by increasing the concentration of NaCl in the equilibration buffer as follows: 0–30 min, no salt; 30–90 min, a gradient from 0 to 1.0 m NaCl; 90–110 min, 1 mNaCl. The flow rate was 1.0 ml/min. All HPLC analyses were conducted using a Varian 9012 inert solvent delivery system equipped with a polychrome 9065 diode array detector. Peptides were monitored at 254 mm. Fractions 71–76 were pooled and further purified by reverse phase HPLC on a C18 column (Vydac) under ion-pairing conditions. The column was equilibrated with 10 mm triethylammonium acetate (pH 7.0) for 10 min. After a 5-min wash with HPLC-grade water, the peptide(s) were eluted by increasing the concentration of acetonitrile in water from 0 to 30%. The peptides were monitored at 254 nm. Fractions corresponding to peaks I (residues 67–82) and II (residues 90–102) were pooled individually and subjected to amino-terminal amino acid sequencing using the Edman degradation reaction (30.Edman P. Begg G. Eur. J. Biochem. 1967; 1: 80-91Crossref PubMed Scopus (2416) Google Scholar) on an Applied Biosystems 470A gas phase sequencer at the W. M. Keck Foundation Biotechnology Resource Laboratory, Yale University, New Haven, CT. The resulting phenylthiohydantoin derivatives were analyzed using an on-line Applied Biosystems model 470A microbore HPLC. λ Int and its close relatives are unusual in being heterobivalent DNA-binding proteins. A short amino-terminal domain, residues 1–64, is responsible for binding to “arm-type” DNA sequences distant from the core-type DNA sequences where DNA strands are cleaved, exchanged, and religated. All of the operations on core-type DNA, including binding, are governed by the carboxyl-terminal portion of Int (residues 65–356). This autonomously functioning protein, called C65, has been cloned and purified (20.Tirumalai R.S. Kwon H. Cardente E. Ellenberger T. Landy A. J. Mol. Biol. 1998; 279: 513-527Crossref PubMed Scopus (45) Google Scholar) and, unless noted otherwise, is considered exclusively in this paper. Its activity on core-type DNA is not only undiminished but is actually enhanced by separation from the amino-terminal domain (31.Sarkar D. Radman-Livaja M. Landy A. EMBO J. 2000; 20: 1203-1212Crossref Scopus (42) Google Scholar). To monitor the efficiency of Int C65 binding and DNA cleavage, we took advantage of a suicide core site (32.Nunes-Düby S.E. Matsumoto L. Landy A. Cell. 1987; 50: 779-788Abstract Full Text PDF PubMed Scopus (185) Google Scholar). As diagrammed in Fig. 2, these substrates contain a 3′ terminus three bases from the scissile phosphate. When Int cleaves this substrate (via formation of a covalent 3′-phosphotyrosine linkage) it generates a 3-base oligonucleotide that diffuses away from theatt site DNA. Loss of the oligonucleotide removes the 5′-OH nucleophile that would otherwise attack the phosphotyrosine bond to reform the phosphodiester DNA linkage and release Int. In the absence of the 5′-OH nucleophile, the phosphotyrosine linkage is stable, and the covalent complex is readily monitored or isolated by gel electrophoresis in SDS-polyacrylamide. In the experiments reported here we used short synthetic oligonucleotides (22/23- or 30/34-mers) containing a “half-att site.” This consists of a single 8-bp core-type Int-binding site and a portion of the 7-bp overlap region. The cleaved strands (top strands in all of the figures) contained 3 bases of the overlap region, which are lost upon Int cleavage. The bottom strands contained either 4 or the full 7 bases of the overlap region. We have not observed any differences in binding or cleavage efficiency between substrates with 4–7 bases in the bottom strand of the overlap region nor between substrates containing 12versus 20 bp preceding the Int-binding site (data not shown). We constructed 11 half-att site substrates, each containing a 4-thioT substitution at a different position and labeled with32P at the 5′ terminus of the top strands. Because we are incorporating a thio analog, and in some cases substituting a thymine for the canonical base, we tested each substrate for its ability to be cleaved by wild-type Int and form a suicide covalent complex (as diagrammed in Fig. 2) which is seen as a band with slower electrophoretic mobility in SDS-polyacrylamide than the free DNA. Most of the substrates were very efficient at forming covalent complex (Fig. 3 A). (A lane showing cleavage of the unsubstituted att site was not included in the gel shown in Fig. 3 but in similar experiments its efficiency is approximately the same as att sites T3, B4, B6, T8, T9, T11, B11, T12, and B12). The B5 att site was quite depressed for Int cleavage, and the T4 att site was not cleaved at all by Int. To test for photo-cross-linking efficiency, each of the attsites was incubated with Int C65 Y342F (C65F), a mutant in which the active site nucleophile, tyrosine 342, has been substituted by phenylalanine. We have shown previously that this mutant is completely defective in DNA cleavage as assayed by topoisomerase activity, covalent complex formation with suicide substrates, and resolution of Holliday junction recombination intermediates (14.Pargellis C.A. Nunes-Düby S.E. Moitoso de Vargas L. Landy A. J. Biol. Chem. 1988; 263: 7678-7685Abstract Full Text PDF PubMed Google Scholar). The C65F-att site reactions were incubated for 20 min at 25 °C and then irradiated at 4 °C on a pre-chilled porcelain plate with 366 nm UV light (see “Experimental Procedures”). The formation of photo-induced Int C65F covalent adducts was assayed by gel electrophoresis alongside of the wild-type covalent suicide-cleavage complexes (Fig. 3 A). Quantitation of the percent photo-cross-linking is shown in Fig. 3 B along with the position of each 4-thioT in the half-att site substrates. Those att sites such as B5 and T4, for which the 4-thioT substitution clearly interfered with Int cleavage (and/or binding), would not be expected to yield efficient photo-cross-linking, and indeed they are very poor. Their observed level of cross-linking is approximately equal to that of the unsubstituted core-type site (0.5%) (data not shown). Whereas these two substitutions may be poor photo-cross-linking substrates because the 4-thioTs intrude too severely into the Int-binding space (to the point of preventing Int cleavage), there will be other substitutions that are poor photo-cross-linking substrates because the thio group is too far from the bound Int (or from a reactive residue within Int). In this category of att sites, which are cleaved efficiently by Int but not efficiently photo-cross-linked (<3%), we find the B6, T8, T9, B11, and B12 substitutions. The group of att sites yielding intermediate photo-cross-linking efficiencies (∼5%) consists of the B4, T11, and T12 substitutions. The T3 substitution, which consistently yielded photo-cross-linking efficiencies of ∼20%, was clearly the best of those we tested. It should be noted that the level of cross-linking of even the poorest 4-thioT substrates is still well above the 0.5% that is obtained with unsubstituted attsites (data not shown). The high efficiency of ph