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• W1972130106 abstract "We investigated which features of the substrate specificity of human immunodeficiency virus type 1 (HIV-1) integrase could be assigned to the central domain of the 288-residue HIV-1 integrase protein, composed of amino acids 50–212. This domain contains the active site and shares structural homology with a large family of polynucleotidyl transferases. Using model substrates with defined alterations in critical features we found that this domain alone is sufficient for recognition of: 1) the phylogenetically conserved CA/TG base pairs near the viral DNA end; 2) the 5′-terminal dinucleotide that is left unpaired after end processing; and 3) target DNA flanking the site of joining. Future efforts aimed at identifying specific amino acids involved in recognition of these key substrate features can now be targeted at this domain. We investigated which features of the substrate specificity of human immunodeficiency virus type 1 (HIV-1) integrase could be assigned to the central domain of the 288-residue HIV-1 integrase protein, composed of amino acids 50–212. This domain contains the active site and shares structural homology with a large family of polynucleotidyl transferases. Using model substrates with defined alterations in critical features we found that this domain alone is sufficient for recognition of: 1) the phylogenetically conserved CA/TG base pairs near the viral DNA end; 2) the 5′-terminal dinucleotide that is left unpaired after end processing; and 3) target DNA flanking the site of joining. Future efforts aimed at identifying specific amino acids involved in recognition of these key substrate features can now be targeted at this domain. Integration of a double-stranded DNA copy of the retroviral genome into a host cell chromosome is essential for viral replication. Genetic and biochemical studies have shown that retroviralintegration requires two viral components, integrase and the U3 and U5 att sites, which are phylogenetically conserved sequences at the ends of viral DNA. Analysis in vivo and in vitrohas revealed that integrase processively catalyzes two chemical steps. In 3′-end processing, integrase cleaves two nucleotides from the 3′-ends of the double-stranded viral DNA, leaving a phylogenetically invariant CA dinucleotide sequence at the 3′-termini of the recessed viral DNA ends. In the second chemical step, integrase catalyzes the joining of the two 3′-recessed viral DNA ends to sites on opposite strands of a target DNA, separated by 5 base pairs (bps) 1The abbreviations used are: bp(s), base pair(s); HIV-1, human immunodeficiency virus type 1; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. in the case of HIV-1. The gaps that flank this product of the joining reaction are repaired by a pathway that remains uncharacterized. The chemical reactions catalyzed by integrase can be studied in vitro using model oligonucleotide substrates and purified recombinant protein. We evaluated the specificity of the isolated core domain of HIV-1 integrase. A minimal catalytically active peptide has been identified by deletion analysis, comprised of amino acids 50–186 (1Bushman F.D. Engelman A. Palmer I. Wingfield R.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3428-3432Crossref PubMed Scopus (333) Google Scholar). To ensure reasonable dynamic range in our assays, we used a slightly larger, more active polypeptide, consisting of amino acids 50–212 (1Bushman F.D. Engelman A. Palmer I. Wingfield R.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3428-3432Crossref PubMed Scopus (333) Google Scholar). The three-dimensional structure of this domain has been solved, and it reveals striking structural homology to a larger family of polynucleotidyl transferases (2Dyda F. Hickman A.B. Jenkins T.M. Engelman A. Craigie R. Davies D.R. Science. 1994; 266: 1981-1986Crossref PubMed Scopus (725) Google Scholar). This domain contains the phylogenetically conserved DD35E motif that defines the active site (3Bujacz G. Jaskolski M. Alexandratos J. Wlodawer A. Merkel G. Katz R.A. Skalka A.M. Structure. 1996; 4: 89-96Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 4Leavitt A.D. Shiue L. Varmus H.E. J. Biol. Chem. 1993; 268: 2113-2119Abstract Full Text PDF PubMed Google Scholar, 5Drelich M. Wilhelm R. Mous J. Virology. 1992; 188: 459-468Crossref PubMed Scopus (174) Google Scholar, 6Engelman A. Craigie R. J. Virol. 1992; 66: 6361-6369Crossref PubMed Google Scholar, 7Kulkosky J. Jones K.S. Katz R.A. Mack A.M.S. Mol. Cell. Biol. 1992; 12: 2331-2338Crossref PubMed Scopus (523) Google Scholar, 8van Gent D.C. Groeneger A.A. Plasterk R.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9598-9602Crossref PubMed Scopus (182) Google Scholar). Using model substrates with defined alterations in features known to be critical for the catalytic specificity of full-length integrase, we assessed the ability of the isolated core domain to distinguish between altered and wild-type substrates. Integrase can carry out a concerted cleavage-ligation reaction, termed disintegration, on a model substrate that mimics the product of integration of a single viral DNA end, yielding a free cleaved viral DNA end and a ligated target DNA strand (9Chow S.A. Vincent K.A. Ellison V. Brown P.O. Science. 1992; 255: 723-726Crossref PubMed Scopus (365) Google Scholar). A divalent metal ion is required for catalysis of disintegration as well as for end processing and joining (9Chow S.A. Vincent K.A. Ellison V. Brown P.O. Science. 1992; 255: 723-726Crossref PubMed Scopus (365) Google Scholar, 10Brown P.O. Bowerman B. Varmus H.E. Bishop J.M. Cell. 1987; 49: 347-356Abstract Full Text PDF PubMed Scopus (331) Google Scholar, 11Craigie R. Fujiwara T. Bushman F. Cell. 1990; 62: 829-837Abstract Full Text PDF PubMed Scopus (331) Google Scholar). Disintegration substrates have proven extremely useful in studying viral DNA recognition (1Bushman F.D. Engelman A. Palmer I. Wingfield R.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3428-3432Crossref PubMed Scopus (333) Google Scholar, 12Chow S.A. Brown P.O. J. Virol. 1994; 68: 3896-3907Crossref PubMed Google Scholar, 13Chow S.A. Brown P.O. J. Virol. 1994; 68: 7869-7878Crossref PubMed Google Scholar, 14Vincent K.A. Ellison V. Chow S.A. Brown P.O. J. Virol. 1993; 67: 425-437Crossref PubMed Google Scholar), target DNA recognition (12Chow S.A. Brown P.O. J. Virol. 1994; 68: 3896-3907Crossref PubMed Google Scholar), catalytic requirements 2J. L. Gerton, D. Herschlag, and P. O. Brown, manuscript in preparation. (12Chow S.A. Brown P.O. J. Virol. 1994; 68: 3896-3907Crossref PubMed Google Scholar), and kinetic properties 3J. L. Gerton, S. Ohgi, M. Olsen, J. DeRisi, and P. O. Brown, manuscript in preparation. (12Chow S.A. Brown P.O. J. Virol. 1994; 68: 3896-3907Crossref PubMed Google Scholar) of full-length and mutant integrases. The disintegration substrate allows one to analyze the catalytic properties of mutant derivatives of integrase, such as the core domain, which have defects in requirements for the forward reaction. The orientation of viral and target DNA with respect to the active site is similar for disintegration and joining.2 Therefore, specificity observed in the context of a disintegration substrate is likely to follow the same rules that govern specificity in the integration reaction; indeed, similar catalytic specificity for disintegration and joining has been observed (12Chow S.A. Brown P.O. J. Virol. 1994; 68: 3896-3907Crossref PubMed Google Scholar, 13Chow S.A. Brown P.O. J. Virol. 1994; 68: 7869-7878Crossref PubMed Google Scholar, 14Vincent K.A. Ellison V. Chow S.A. Brown P.O. J. Virol. 1993; 67: 425-437Crossref PubMed Google Scholar). We monitored the sensitivity of the core domain to alterations in the following substrate features: the phylogenetically conserved CA/TG bps located immediately internal to the processing site at the viral DNA end, the 5′-dinucleotide left unpaired at the viral DNA end after 3′-end processing, and target DNA flanking the joining site. The infectivity of retroviruses containing mutations in the CA/TG bps is reduced 10−5 in vivo (15Roth M.J. Schwartzberg P.L. Goff S.P. Cell. 1989; 58: 47-54Abstract Full Text PDF PubMed Scopus (188) Google Scholar); mutations in these bps can also seriously compromise activity of model substrates in vitro (11Craigie R. Fujiwara T. Bushman F. Cell. 1990; 62: 829-837Abstract Full Text PDF PubMed Scopus (331) Google Scholar, 16Sherman P.A. Fyfe J.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5119-5123Crossref PubMed Scopus (303) Google Scholar, 17Bushman F.D. Craigie R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1339-1343Crossref PubMed Scopus (355) Google Scholar, 18LaFemina R.L. Callahan P.L. Cordingly M.G. J. Virol. 1991; 65: 5624-5630Crossref PubMed Google Scholar, 19Leavitt A.D. Rose R.B. Varmus H.E. J. Virol. 1992; 66: 2359-2368Crossref PubMed Google Scholar, 20Sherman P.A. Dickson M.L. Fyfe J.A. J. Virol. 1992; 66: 3593-3601Crossref PubMed Google Scholar, 21van den Ent F. Vink C. Plasterk R.H.A. J. Virol. 1994; 68: 7825-7832Crossref PubMed Google Scholar, 22Scottoline B.P. Chow S. Ellison V. Brown P.O. Genes Dev. 1997; 11: 371-382Crossref PubMed Scopus (76) Google Scholar). The 5′-terminal dinucleotide on the unprocessed strand increases the stability of integrase-viral DNA end complexes formed in vitro and enhances processivity between the end processing and joining steps (23Ellison V. Brown P.O. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7316-7320Crossref PubMed Scopus (152) Google Scholar). Although most sites can be used at some level as integration targets, HIV-1 integrase has target site preferences; these preferences are determined both by DNA structure (24Pryciak P.M. Varmus H.E. Cell. 1992; 69: 769-780Abstract Full Text PDF PubMed Scopus (235) Google Scholar, 25Pruss D. Bushman F.D. Wolffe A.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5913-5917Crossref PubMed Scopus (208) Google Scholar, 26Pruss D. Reeves R. Bushman F.D. Wolffe A.P. J. Biol. Chem. 1994; 269: 25031-25041Abstract Full Text PDF PubMed Google Scholar) and sequence (27Bor Y.C. Bushman F.D. Orgel L.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10334-10338Crossref PubMed Scopus (59) Google Scholar). Domain swaps among integrases from different retroviral species indicate that the core domain, in large part, determines target site preferences as well as specificity for the species-specific features of the viral DNA end (28Katzman M. Sudol M. J. Virol. 1995; 69: 5687-5696Crossref PubMed Google Scholar, 29Pahl A. Flugel R.M. J. Biol. Chem. 1995; 270: 2957-2966Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 30Shibagaki Y. Chow S.A. J. Biol. Chem. 1997; 272: 8361-8369Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). These experiments, however, do not allow the identification of the integrase domain that recognizes the conserved features of the viral DNA end. The crystal structure of the catalytic core domain of HIV-1 integrase has been solved to 2.5 Å (2Dyda F. Hickman A.B. Jenkins T.M. Engelman A. Craigie R. Davies D.R. Science. 1994; 266: 1981-1986Crossref PubMed Scopus (725) Google Scholar). Crystallization was made possible by the discovery of a mutation, F185K, which enhances solubility but does not affect the in vitro biochemical properties of integrase, except to lower the K d of the constitutive dimer formed in vitro (31Jenkins T.M. Hickman A.B. Dyda F. Ghirlando R. Davies D.R. Craigie R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6057-6061Crossref PubMed Scopus (141) Google Scholar, 32Jenkins T.M. Engelman A. Ghirlando R. Craigie R. J. Biol. Chem. 1996; 271: 7712-7718Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). The crystal structure reveals an extensive hydrophobic interface between two monomers. The results presented here on substrate specificity, together with the biophysical and structural information available for the core domain, place new constraints on models of integrase-substrate interactions within the multimeric complex that catalyzes end processing and joining. Full-length HIV-1 integrase was overexpressed in Escherichia coli using the T7 polymerase promoter system and purified as described previously (14Vincent K.A. Ellison V. Chow S.A. Brown P.O. J. Virol. 1993; 67: 425-437Crossref PubMed Google Scholar). A deletion construct containing amino acids 50–212 of HIV-1 integrase in the expression strain BL21 was a gift from Tim Jenkins. The gene contains a mutation, F185K, which increases the solubility of the protein. In vitro characterization of full-length integrase containing this mutation has demonstrated that its biochemical properties are virtually indistinguishable from the wild-type protein, with the exception that the mutation appears to allow the core domain protein to form a tighter dimer in solution (31Jenkins T.M. Hickman A.B. Dyda F. Ghirlando R. Davies D.R. Craigie R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6057-6061Crossref PubMed Scopus (141) Google Scholar). The construct encoding the core domain also codes for six histidines appended to the amino terminus. The hexahistidine-tagged 50–212 (F185K) polypeptide was expressed in the same manner as full-length integrase but purified using Ni-affinity chromatography. The thawed bacterial pellet from a 2-liter culture grown in Luria broth (LB) was washed with 20 mm HEPES, pH 7.5, 1 mm EDTA, then resuspended in 40 ml of lysis buffer containing 20 mmTris-HCl, pH 8.0, 0.1 mm EDTA, 2 mmβ-mercaptoethanol, 0.5 m NaCl, and 2 mg/ml lysozyme. After 30 min on ice, the solution was sonicated with five 30-s pulses. After sonication, the cells were centrifuged at 18,000 rpm in an SS-34 rotor for 45 min at 4 °C. The pellet was resuspended in 50 ml of 20 mm Tris-HCl, pH 8.0, 1 m NaCl, 2 mmβ-mercaptoethanol (TNM) containing 5 mm imidazole, then processed 30 times in a Dounce homogenizer. This high salt extract was stirred at 4 °C for 1 h, then centrifuged at 28,000 rpm in an SW28 rotor for 1 h at 4 °C. The supernatant was loaded by gravity onto a 1-ml column of Ni2+-nitrilotriacetic acid agarose (Ni-NTA resin, Qiagen) equilibrated in TNM containing 5 mm imidazole. The column was then washed with 20 column volumes of TNM containing 5 mm imidazole, then 20 column volumes of TNM containing 40 mm imidazole. Protein was eluted using a gradient of 40–600 mm imidazole in TNM. Fractions were pooled based on the Bradford assay and catalytic activity in disintegration assays. The pooled fractions were passed over a second 1-ml Ni-NTA column to eliminate contaminating nucleases. The pooled fractions were diluted 1:1 with 20 mm HEPES, pH 7.5, 10 mm dithiothreitol, and 10 mm CHAPS and incubated with thrombin at a concentration of 40 NIH units/mg integrase protein for 3 h at 16 °C, to cleave off the amino-terminal hexahistidine tag. Diisopropyl fluorophosphate was added to 1 μg/ml to inactivate the thrombin. The thrombin-digested material was loaded onto a 1-ml DEAE-Sepharose (Pharmacia Biotech Inc.) column. This column was washed with 10 column volumes of buffer (HCDG) containing 50 mm HEPES, pH 7.5, 10 mm CHAPS, 2 mmdithiothreitol, 10% glycerol with 100 mm NaCl. One ml each of HCDG containing 250 mm NaCl, 500 mm NaCl, 750 mm NaCl, or 1 m NaCl was added sequentially, and 1-ml fractions were collected. The 50–212 (F185K) polypeptide eluted in the 500, 750, and 1,000 mm NaCl steps. Aliquots were frozen in liquid nitrogen before storage at −80 °C. Protein concentration was determined by the Bradford assay. The fraction eluting with 750 mm NaCl was used for all experiments. The disintegration substrates used were either of the “dumbbell” type or the “Y-mer” type. Both have a branched structure, mimicking one viral end joined to target DNA (9Chow S.A. Vincent K.A. Ellison V. Brown P.O. Science. 1992; 255: 723-726Crossref PubMed Scopus (365) Google Scholar). The standard Y-mer-type substrate is composed of four oligonucleotides annealed together. The resulting structure has 19 bps representing viral DNA and 30 bps representing target DNA. Dumbbell refers to substrates in which one oligonucleotide folds upon itself to form the branched structure. The standard dumbbell substrate is a 40-mer (dby4), which folds to produce a substrate that contains 5 bps representing viral DNA, joined to 10 bps representing target DNA. All oligonucleotides were purchased from Operon Technologies, Inc. (Emeryville, CA) and were purified by electrophoresis through a 10 or 15% denaturing polyacrylamide gel before use in the construction of substrates for activity assays. Oligonucleotides were labeled at the 5′-end using T4 polynucleotide kinase (New England Biolabs) and [γ-32P]ATP (Amersham, 3,000 Ci/mmol). Unincorporated radioactive nucleotides were removed from the labeled oligonucleotide by centrifugation through 1-ml columns of Sephadex G-15 (Sigma). Preparation of the Y-mer substrate and structurally related substrates for disintegration assays was done as follows. The 5′-end-labeled oligonucleotide was added to a 3-fold molar excess of the other three oligonucleotides composing the Y-mer. Oligonucleotides in a solution containing 10 mm Tris-HCl, pH 8.0, 1 mm EDTA, and 50 mm NaCl (TEN) were heated to 90 °C and allowed to cool slowly to room temperature. Ficoll loading buffer was added to achieve a final concentration of 2% Ficoll 400, 1 mm EDTA, 0.025% bromphenol blue, and 0.025% xylene cyanol, and the sample was then electrophoresed on a 10% nondenaturing polyacrylamide gel 16 h at 250 volts. The wet gel was autoradiographed, and the band corresponding to the completely annealed substrate was excised and eluted overnight in 0.5m ammonium acetate and 10 mm magnesium acetate. The supernatant fluid was concentrated in a Centricon-10 (Amicon) by centrifugation at 4 °C in an SS-34 rotor at 6,500 rpm. After concentration of the eluate from the gel slice, 2.5 ml of TEN was added, and the Centricon-10 was centrifuged another 2 h. The concentration of substrate was calculated based on the specific activity of the labeled oligonucleotide. The dumbbell and its related counterparts were prepared by heating the oligonucleotide in TEN to 90 °C and then slowly cooling to room temperature. Oligonucleotide sequences used to make the U5 viral DNA end Y-mer substrates were described in Chow et al. (9Chow S.A. Vincent K.A. Ellison V. Brown P.O. Science. 1992; 255: 723-726Crossref PubMed Scopus (365) Google Scholar). The additional oligonucleotide sequences used to make the U3 viral DNA end Y-mer disintegration substrates were as follows. 5′-ATGTGAATTAGCCCTTCCAGGCTGCAGGTCGAC-3′. 5′-ACT GGA AGG GCT AAT TCA CAT-3′. Sequences of oligonucleotides used to test the dependence of the core domain on target DNA were as follows. 5′-ACTGCTAGTTCTAGCAGGCCCTTGGGCCGGCGCTTGCGCC-3′. 5′-ACTGCTAGTTCTAGCAGGCCCCATTTGGGGCCGGCGCTTTTAAGCGCC-3′. 5′-ACTGCTAGTTCTAGCAGGCCCCAGGTTGGTGGGGCCGGCGCTTGCTTGCAGCGCC-3′. 5′-ACTGCTAGTTCTAGCAGGGCCCCAGGTCTTGACCTGGGGCCGGCGCTTGCGTTTACGCAAGCGCC-3′. 5′-ACTGCTAGTTCTAGCAGGCTGCAGGTCGACTTGTCGACCTGCAGCCCAAGCTTGCGTTGCTGTTCAGCAACGCAAGCTTG-3′. 5′-ACTGCTAGAGATTTTCCACATTTATGTGGAAAATCTCTAGCAGGCCCTTGGGCCGGCGCTTGCGCC-3′. 5′-ACTGCTAGTTCTAGCAGGCTGCAGGTCGACTTGTCGACCTGCAGCCCGTTCG-3′. 5′-ACTGCTAGTTCTAGCAGCTTGCCAAGCTTGCGTTGCTTGCAACGCAAGCTTG-3′. All disintegration reactions were performed in solution containing 20 mm HEPES, pH 7.5, 10–20 mmdithiothreitol, 10 mm MnCl2, and 0.05% Nonidet P-40. The NaCl concentration was kept at or below 10 mmexcept where noted in the figure legend. Reactions were stopped by the addition of an equal volume of formamide loading buffer (95% formamide, 50 mm EDTA, 0.1% bromphenol blue, and 0.1% xylene cyanol). Reactions were heated to 90 °C for 2–3 min before loading onto a 20% denaturing polyacrylamide gel. Quantitation of products was carried out with a Molecular Dynamics PhosphorImager. All reactions (except turnover experiments) were performed with 150 nm integrase and 20 nm substrate and were incubated for 30 min at 37 °C. For turnover experiments, 100 or 200 nm integrase was incubated with 1,000 nm Y-mer at 37 °C. Aliquots were removed from the reaction at the indicated time intervals and stopped by the addition of an equal volume of formamide loading buffer. The concentrations of integrase cited throughout this report refer to protomers. Y-mer disintegration substrates with altered viral DNA portions were constructed to investigate the sensitivity of the HIV-1 catalytic core domain to gross structural alterations in the viral DNA. Alterations made to disintegration substrates included: 1) replacement of the viral DNA portion with single-stranded viral DNA (Fig. 1, lanes 7–9); 2) replacement of the viral DNA with a single adenosine nucleotide (Fig.1, lanes 4–6); 3) changing the phylogenetically conserved CA/TG bps of the viral DNA end to TC/GA (Fig. 1, lanes 10–12). A substrate in which the CA/TG bps were instead changed to GT/AC was also tested, and the results were qualitatively similar to those obtained for the CA/TG to TC/GA variant (data not shown). The core domain was clearly able to distinguish these grossly altered viral DNA ends from a wild-type viral DNA end (Fig. 1, lanes 1–3). Under these reaction conditions, the core domain was more sensitive to these gross changes in the viral DNA end than was full-length integrase. To determine whether the core domain could specifically recognize the conserved CA/TG bps of the viral DNA end, we measured the catalytic activity of the core domain on Y-mer disintegration substrates with base substitutions in the phylogenetically conserved A/T bp (Fig.2 A) or C/G bp (Fig.2 B). Each substrate contained one of three types of alterations: 1) two complementary mutant bases substituted for the wild-type bp; 2) two noncomplementary mutant bases substituted for the wild-type bp; or 3) one mutant base mispaired with one wild-type base substituted for the wild-type bp. This approach allowed the effects of structure (matched or mismatched) to be distinguished from the effects of specific base substitutions. The amounts of product formed in reactions with wild-type substrate are shown in the two far right lanes in Fig. 2, indicated by A/T in panel A and C/G in panel B. The pair of bases substituted for the A/T bp (panel A) or the C/G bp (panel B) is specified below the column. For this analysis, 150 nmintegrase and 20 nm substrate were incubated at 37 °C for 30 min. At least two nucleotide changes were tested at each of the four positions in the viral CA/TG bps. Changing the wild-type A/T bp to the complementary mutant bp G/C or T/A resulted in a 21- or 14-fold decrease, respectively, in product formed from a disintegration substrate by the core domain. Similarly, changing the C/G bp to G/C or T/A led to a 20- or 15-fold reduction, respectively, in product formed from a disintegration substrate by the core domain. Thus, the core domain can recognize these conserved bases in the viral DNA end. Indeed, the core domain was more sensitive to alterations of these bps than was full-length integrase, which produced roughly 2-fold less product from substrates with the wild-type A/T bp changed to G/C or T/A and roughly 3-fold less product from disintegration substrates in which the wild-type C/G bp was changed to G/C or T/A. Similar reductions in the disintegration activity of the core domain were observed when these bps were replaced by noncomplementary alternative base pairs. Replacing the wild-type A/T bp with T/C or G/A resulted in a 4- or 20-fold reduction, respectively, in product formed from a disintegration substrate. Replacing the wild-type C/G bp with G/A or T/C led to a 50-fold and 6-fold reduction, respectively, in product formed from a disintegration substrate by the core domain. Although full-length integrase also showed reduced activity on these mutant substrates, the effect of the mutations was less severe (2–3-fold less product than on a wild-type substrate). Substrates in which a wild-type base was paired with a noncomplementary base, generating a mismatch, were, in general, worse substrates than the wild-type substrate for both full-length integrase and the core domain. Once again, the viral DNA end mutations had a more severe effect on the activity of the core domain than on full-length integrase. The core domain produced 4–100-fold less product from mismatched substrates in which the A, C, or G bases were altered and mispaired with a wild-type base (the A/T bp was changed to G/T and T/T, the C/G bp was changed to G/G, T/G, C/C, and C/A) than on a wild-type substrate. For full-length integrase, identical mutations in the substrate resulted in up to an 8-fold decrease in product. One subset of mismatched mutant substrates actually proved to be slightly better substrates than wild-type substrates. The core domain and full-length integrase both generated slightly more product from substrates containing a wild-type A base mispaired with a C (panel A,A/C) or A base (panel A, A/A) than from a wild-type substrate (panel A, A/T). It has been demonstrated previously that incorporating mismatches at the A/T position in a viral DNA end substrate can facilitate end processing in vitro, strongly suggesting that fraying of the terminal bps of the viral DNA precedes cleavage (22Scottoline B.P. Chow S. Ellison V. Brown P.O. Genes Dev. 1997; 11: 371-382Crossref PubMed Scopus (76) Google Scholar). Mismatches at this position resulting from substitutions for the thymine residue in disintegration substrates lower the energetic barrier to fraying, enhancing disintegration of these substrates by both the core domain and full-length integrase. In contrast, mismatches at the A/T bp caused by substitutions for the adenosine residue did not enhance disintegration. Presumably, the favorable effect of the fraying did not outweigh the deleterious effect of the lack of base-specific interactions with the adenosine residue. In all cases (with the exception of substrates containing a wild-type A base mispaired with a mutant base), the mutant disintegration substrates were poorer substrates than the wild-type substrate for the core domain protein, indicating that features of integrase capable of recognizing the phylogenetically invariant CA/TG bps are contained in the core domain. Furthermore, the core domain was more sensitive to alterations in the conserved A/T and C/G bps than was full-length integrase, despite their similar activity on wild-type substrates (either a U5 or U3 viral DNA end; data shown for the U5 end only). The hypersensitivity of the core domain to mutations in these bps may be a result of the loss of the nonspecific DNA binding activity of the COOH terminus (33Engelman A. Hickman A. Craigie R. J. Virol. 1994; 68: 5911-5917Crossref PubMed Google Scholar, 34Puras-Lutzke R.A. Vink C. Plasterk R.H.A. Nucleic Acids Res. 1994; 22: 4125-4131Crossref PubMed Scopus (173) Google Scholar, 35Vink C. Oude Groeneger A.M.M. Plasterk R.H.A. Nucleic Acids Res. 1993; 21: 1419-1425Crossref PubMed Scopus (247) Google Scholar), making the core domain more dependent on the remaining specific contacts with the CA/TG bps. The disintegration activity of the core domain of HIV-1 integrase has been reported to be lower than that of full-length integrase (1Bushman F.D. Engelman A. Palmer I. Wingfield R.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3428-3432Crossref PubMed Scopus (333) Google Scholar). We attribute this discrepancy with our results to the different reaction conditions used. NaCl and MnCl2 titrations revealed that the isolated core domain was extremely sensitive to the ionic milieu of the reaction. Concentrations of NaCl or MnCl2 above 10 mm caused a precipitous drop in the activity of the core domain (data not shown). Full-length HIV-1 integrase, in contrast, could catalyze disintegration under reaction conditions containing up to 100 mm NaCl or 100 mm MnCl2. Electrostatic interactions thus appear to play a critical role in the residual DNA binding activity of the core domain. When disintegration reactions were carried out in solutions containing 10 mm NaCl or less, the core domain often produced slightly more product than full-length integrase, under standard conditions of enzyme excess. This observation prompted the comparison of turnover in disintegration reactions catalyzed by the two proteins. The core domain can turn over faster than full-length integrase in reactions using a wild-type Y-mer disintegration substrate (Fig.3); the k cat of the core domain was 1.5 h−1, whereas the k cat of full-length integrase was 0.26 h−1, under reaction conditions favorable to the activity of the core domain. Aliquots were taken at indicated time intervals from reactions containing 1,000 nm wild-type disintegration substrate and 200 nm protein. Each point plotted in Figs. 3and 4 represents the average of two independent determinations. The apparent difference in activity may be an underestimate because the concentration of NaCl in these reactions (16 mm) was sufficient to inhibit partially the activity of the core domain but not that of full-length integrase. Qualitatively similar results were obtained in a similar experiment carried out with 100 nm protein and 1,000 nm substrate (data not shown). (The disintegration substrate was stored in 50 mmNaCl to prevent dissociation of the strands; the large volume of substrate added in these reactions resulted in the relatively “high” concentration of NaCl.)Figure 4Turnover rate in disintegration reactions catalyzed by full-length HIV-1 integrase or the core domain is increased in the absence of the 5′-dinucleotide of the viral DNA end. 1,000 nm Y-mer disintegration substrate was incubated with 200 nm core domain or full-length integrase at 37 °C. Aliquots were removed at 10, 20, 30, 45, 60, 90, 120, 180, 240, and 300 min. Duplicate samples were quantitated using a PhosphorImager and averaged. The NaCl concentration in the reactions wa" @default.
• W1972130106 created "2016-06-24" @default.
• W1972130106 creator A5016119539 @default.
• W1972130106 creator A5065906655 @default.
• W1972130106 date "1997-10-01" @default.
• W1972130106 modified "2023-09-29" @default.
• W1972130106 title "The Core Domain of HIV-1 Integrase Recognizes Key Features of Its DNA Substrates" @default.
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