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• W1978523876 abstract "We have reconstituted concerted human immunodeficiency virus type 1 (HIV-1) integration with specially designed mini-donor DNA, a supercoiled plasmid acceptor, purified bacterial-derived HIV-1 integrase (IN), and host HMG-I(Y) protein (Hindmarsh, P., Ridky, T., Reeves, R., Andrake, M., Skalka, A. M., and Leis, J. (1999) J. Virol. 73, 2994–3003). Integration in this system is dependent upon the mini donor DNA having IN recognition sequences at both ends and the reaction products have all of the features associated with integration of viral DNA in vivo. Using this system, we explored the relationship between the HIV-1 U3 and U5 IN recognition sequences by analyzing substrates that contain either two U3 or two U5 terminal sequences. Both substrates caused severe defects to integration but with different effects on the mechanism indicating that the U3 and the U5 sequences are both required for concerted DNA integration. We have also used the reconstituted system to compare the mechanism of integration catalyzed by HIV-1 to that of avian sarcoma virus by analyzing the effect of defined mutations introduced into U3 or U5 ends of the respective wild type DNA substrates. Despite sequence differences between avian sarcoma virus and HIV-1 IN and their recognition sequences, the consequences of analogous base pair substitutions at the same relative positions of the respective IN recognition sequences were very similar. This highlights the common mechanism of integration shared by these two different viruses. We have reconstituted concerted human immunodeficiency virus type 1 (HIV-1) integration with specially designed mini-donor DNA, a supercoiled plasmid acceptor, purified bacterial-derived HIV-1 integrase (IN), and host HMG-I(Y) protein (Hindmarsh, P., Ridky, T., Reeves, R., Andrake, M., Skalka, A. M., and Leis, J. (1999) J. Virol. 73, 2994–3003). Integration in this system is dependent upon the mini donor DNA having IN recognition sequences at both ends and the reaction products have all of the features associated with integration of viral DNA in vivo. Using this system, we explored the relationship between the HIV-1 U3 and U5 IN recognition sequences by analyzing substrates that contain either two U3 or two U5 terminal sequences. Both substrates caused severe defects to integration but with different effects on the mechanism indicating that the U3 and the U5 sequences are both required for concerted DNA integration. We have also used the reconstituted system to compare the mechanism of integration catalyzed by HIV-1 to that of avian sarcoma virus by analyzing the effect of defined mutations introduced into U3 or U5 ends of the respective wild type DNA substrates. Despite sequence differences between avian sarcoma virus and HIV-1 IN and their recognition sequences, the consequences of analogous base pair substitutions at the same relative positions of the respective IN recognition sequences were very similar. This highlights the common mechanism of integration shared by these two different viruses. Integration of retroviral DNA is an obligatory step in viral replication. Integration is catalyzed by the viral encoded enzyme, integrase (IN), 1The abbreviations used are: INintegraseHIVhuman immunodeficiency virusASVavian sarcoma virusLTRlong terminal repeatHMGhigh mobility groupMOPS4-morpholinepropanesulfonic acidRSVRous sarcoma virus which brings the ends of a linear viral DNA together and inserts them into the host chromosome in a concerted reaction (see Ref. 1.Hindmarsh P. Leis J. Microbiol. Mol. Biol. Rev. 1999; 63: 836-843Crossref PubMed Google Scholar for a review). Cell proteins belonging to the HMG-I(Y) family stimulate the reaction (2.Farnet C.M. Bushman F.D. Cell. 1997; 88: 483-492Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar,3.Hindmarsh P. Ridky T. Reeves R. Andrake M. Skalka A.M. Leis J. J. Virol. 1999; 73: 2994-3003Crossref PubMed Google Scholar). The sites of integration are widely distributed in the target DNA. Short duplications of the cell DNA are introduced at the insertion site, the size of which is dictated by IN. For HIV-1 and ASV the size of the duplications are five and six base pairs, respectively. During this process, two base pairs are lost from the ends of the viral LTRs. integrase human immunodeficiency virus avian sarcoma virus long terminal repeat high mobility group 4-morpholinepropanesulfonic acid Rous sarcoma virus The properties of concerted DNA integration have been reconstituted in vitro using purified HIV-1 (3.Hindmarsh P. Ridky T. Reeves R. Andrake M. Skalka A.M. Leis J. J. Virol. 1999; 73: 2994-3003Crossref PubMed Google Scholar, 4.Goodarzi G. Im G.J. Brackmann K. Grandgenett D.P. J. Virol. 1995; 69: 6090-6097Crossref PubMed Google Scholar) or ASV (3.Hindmarsh P. Ridky T. Reeves R. Andrake M. Skalka A.M. Leis J. J. Virol. 1999; 73: 2994-3003Crossref PubMed Google Scholar, 5.Hindmarsh P. Johnson M. Reeves R. Leis J. J. Virol. 2001; 75: 1132-1141Crossref PubMed Scopus (17) Google Scholar, 6.Aiyar A. Hindmarsh P. Skalka A.M. Leis J. J. Virol. 1996; 70: 3571-3580Crossref PubMed Google Scholar, 7.Fitzgerald M.L. Vora A.C. Zeh W.G. Grandgenett D.P. J. Virol. 1992; 66: 6257-6263Crossref PubMed Google Scholar, 8.Vora A.C. Grandgenett D.P. J. Virol. 2001; 75: 3556-3567Crossref PubMed Scopus (31) Google Scholar, 9.Vora A.C. Chiu R. McCord M. Goodarzi G. Stahl S.J. Mueser T.C. Hyde C.C. Grandgenett D.P. J. Biol. Chem. 1997; 272: 23938-23945Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 10.Vora A.C. McCord M. Fitzgerald M.L. Inman R.B. Grandgenett D.P. Nucleic Acids Res. 1994; 22: 4454-4461Crossref PubMed Scopus (55) Google Scholar, 11.Vora A.C. Grandgenett D.P. J. Virol. 1995; 69: 7483-7488Crossref PubMed Google Scholar) IN and MgCl2. The donor DNAs contain only 20 base pairs for HIV-1 or 15 base pairs for ASV derived from the ends of the LTRs, respectively. These viral DNA end sequences correspond to the nearly perfect inverted repeats that define the relationships between the U3 and U5 RNA ends. The inverted repeat for RSV is 12 of 15, while that for HIV-1 is 12 of 20. A comparison of the RSV and HIV-1 IN recognition sequences indicates that they are unique. The only common feature is the presence of a conserved CA dinucleotide at positions 3 and 4 from the terminus. Short oligodeoxynucleotide duplexes representing the ends of HIV-1 U5 LTR are more efficient substrates for IN processing (12.Bushman F.D. Craigie R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1339-1343Crossref PubMed Scopus (355) Google Scholar, 13.Kukolj G. Skalka A.M. Genes Dev. 1995; 9: 2556-2567Crossref PubMed Scopus (38) Google Scholar, 14.Sherman P.A. Dickson M.L. Fyfe J.A. J. Virol. 1992; 66: 3593-3601Crossref PubMed Google Scholar, 15.Cherepanov P. Surratt D. Toelen J. Pluymers W. De Griffith J. Clercq E. Debyser Z. Nucleic Acids Res. 1999; 27: 2202-2210Crossref PubMed Scopus (41) Google Scholar) and strand transfer (15.Cherepanov P. Surratt D. Toelen J. Pluymers W. De Griffith J. Clercq E. Debyser Z. Nucleic Acids Res. 1999; 27: 2202-2210Crossref PubMed Scopus (41) Google Scholar) reactions in vitro than those corresponding to the U3 LTR. In the case of ASV IN, the U3 LTR end is preferred over the U5 LTR end (7.Fitzgerald M.L. Vora A.C. Zeh W.G. Grandgenett D.P. J. Virol. 1992; 66: 6257-6263Crossref PubMed Google Scholar, 9.Vora A.C. Chiu R. McCord M. Goodarzi G. Stahl S.J. Mueser T.C. Hyde C.C. Grandgenett D.P. J. Biol. Chem. 1997; 272: 23938-23945Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 16.Fitzgerald M.L. Vora A.C. Grandgenett D.P. Anal. Biochem. 1991; 196: 19-23Crossref PubMed Scopus (14) Google Scholar). It has also been observed that mutations at the viral U3 LTR end have different effects than those at the U5 end. U3 mutations in ASV reduce integration rate to a greater extent than comparable mutations in U5 (5.Hindmarsh P. Johnson M. Reeves R. Leis J. J. Virol. 2001; 75: 1132-1141Crossref PubMed Scopus (17) Google Scholar, 6.Aiyar A. Hindmarsh P. Skalka A.M. Leis J. J. Virol. 1996; 70: 3571-3580Crossref PubMed Google Scholar). Also, regions critical for integration are very close to the ends of the LTRs, adjacent to and including the conserved CA dinucleotide (5.Hindmarsh P. Johnson M. Reeves R. Leis J. J. Virol. 2001; 75: 1132-1141Crossref PubMed Scopus (17) Google Scholar, 6.Aiyar A. Hindmarsh P. Skalka A.M. Leis J. J. Virol. 1996; 70: 3571-3580Crossref PubMed Google Scholar, 8.Vora A.C. Grandgenett D.P. J. Virol. 2001; 75: 3556-3567Crossref PubMed Scopus (31) Google Scholar,9.Vora A.C. Chiu R. McCord M. Goodarzi G. Stahl S.J. Mueser T.C. Hyde C.C. Grandgenett D.P. J. Biol. Chem. 1997; 272: 23938-23945Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 12.Bushman F.D. Craigie R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1339-1343Crossref PubMed Scopus (355) Google Scholar, 13.Kukolj G. Skalka A.M. Genes Dev. 1995; 9: 2556-2567Crossref PubMed Scopus (38) Google Scholar, 14.Sherman P.A. Dickson M.L. Fyfe J.A. J. Virol. 1992; 66: 3593-3601Crossref PubMed Google Scholar, 15.Cherepanov P. Surratt D. Toelen J. Pluymers W. De Griffith J. Clercq E. Debyser Z. Nucleic Acids Res. 1999; 27: 2202-2210Crossref PubMed Scopus (41) Google Scholar, 17.Katzman M. Katz R.A. Skalka A.M. Leis J. J. Virol. 1989; 63: 5319-5327Crossref PubMed Google Scholar, 18.Ellison V. Brown P.O. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7316-7320Crossref PubMed Scopus (152) Google Scholar, 19.Esposito D. Craigie R. EMBO J. 1998; 17: 5832-5843Crossref PubMed Scopus (263) Google Scholar, 20.Brown H.E. Chen H. Engelman A. J. Virol. 1999; 73: 9011-9020Crossref PubMed Google Scholar). Alteration of these sequences results in retroviral strains deficient in integration. Assays that utilize oligodeoxynucleotide duplexes that represent either U3 or U5 HIV-1 LTR ends have also demonstrated the importance of positions 3–6 to the efficiency of the processing reaction (14.Sherman P.A. Dickson M.L. Fyfe J.A. J. Virol. 1992; 66: 3593-3601Crossref PubMed Google Scholar, 19.Esposito D. Craigie R. EMBO J. 1998; 17: 5832-5843Crossref PubMed Scopus (263) Google Scholar). While an individual LTR end serves as an IN substrate, interactions between IN and both LTR ends determines the mechanism and efficiency of integration in vivo. Therefore, it is important to analyze the effect of mutations in the IN recognition sequences using substrates that contain both LTR termini and are capable of concerted DNA integration. We have used the HIV-1 reconstituted integration system to analyze donor DNAs that contain only U5 or only U3 LTR IN recognition sequences at both ends. Both substrates caused severe defects to integration but with different effects on the mechanism. In addition, a series of mutations were introduced either into the U5 or the U3 HIV-1 IN recognition sequence into and adjacent to the conserved CA dinucleotide of wild type donors. Taken together, these analyses indicated that IN-catalyzed concerted DNA integration requires both U3 and U5 IN recognition sequences in a donor. [α-32P]dCTP (3000 Ci/mmol) was purchased from Amersham Biosciences, Inc. Proteinase K (30 units/mg) and glycogen were from Roche Molecular Biochemicals (Indianapolis, IN). HMG-I(Y) was purified as described by Nissen et al. (21.Nissen M.S. Langan T.A. Reeves R. J. Biol. Chem. 1991; 266: 19945-19952Abstract Full Text PDF PubMed Google Scholar). Vent DNA polymerase (2 units/μl) was from New England Biolabs (Beverly, MA). Oligodeoxyribonucleotides were purchased from Operon (Alameda, CA) and purified by polyacrylamide gel electrophoresis under denaturing conditions. The following oligodeoxyribonucleotides were used in this study: U5(WT), 5′-ACTGCTAGAGATTTTCCACACTGGGCGGAGCCTATG-3′; U5 5GA6, 5′-ACTGGAAGAGATTTTCCACACTGGGCGGAGCCTATG-3′; U5 4CGAT7, 5′-ACTCGATGAGATTTTCCACACTGGGCGGAGCCTATG-3′; U5 3AC4, 5′-ACCCCTAGAGATTTTCCACACTGGGCGGAGCCTATG-3′; U5 del, 5′-CTCATGCACTGCGCGTACACCTGGGCGGAGCCTATG-3′; U5 for U3, 5′-ACTGCTAGAGATTTTCCACACGTTGCCCGGATCCGG-3′; U3(WT), 5′-ACTGGAAGGGCTAATTCACTCGTTGCCCGGATCCGG-3′; U3 5CT6, 5′-ACTGCTAGGGCTAATTCACTCGTTGCCCGGATCCGG-3′; U3 4CCTT7, 5′-ACTCCTTGGGCTAATTCACTCGTTGCCCGGATCCGG-3′; U3 3AC4, 5′-ACACGAAGGGCTAATTCACTCGTTGCCCGGATCCGG-3′; U3 del, 5′-CTCATGCACTGCGCGTACACCGTTGCCCGGATCCGG-3′; U3 for U5, 5′-ACTGGAAGGGCTAATTCACTCTGGGCGGAGCCTATG-3′; U5seq, 5′-AGAATTCGGCGTTGCTGGCGTTTTTCCATA-3′; U3seq, 5′-CTGCCGTCATCGACTTCGAAGGTTCGAATC-3′. The U5 3AC4, U5 5GA6, U5 4CGAT7, and U3 for U5 oligodeoxyribonucleotides were used to prepare HIV-1 donor-concerted DNA integration substrates with mutations in the U5 terminus sequence. In each case, the sequence refers to the 3′-cleaved strand of the U5 LTR IN recognition sequence. The U3 5CT6, 3AC4, 4CCTT7, and U5 for U3 oligodeoxynucleotides were used to prepare comparable donor DNAs with mutations in the U3 IN recognition sequence. The U5seq and U3seq oligodeoxyribonucleotides were used as sequencing primers. The U3seq primer is complementary to plasmid πvx nucleotides 180–151, and the U5seq primer is complementary to plasmid πvx nucleotides 312–341. Escherichia coli DH5α (Invitrogen) and MC1061/P3 (Invitrogen) strains were used for these studies. MC1061/P3 is a derivative of MC1061 containing the male episome, P3, which can be selected for the presence of an encoded Kanr gene. In addition, P3 possesses amp (Am) and tet (f Am) genes, the expression of which can be rescued by the supF amber suppressor tRNA. Under these conditions, MC1061/P3 can be selected for ampicillin, tetracycline, and kanamycin resistance. Plasmid pHHIV2 was used in this study as a template to amplify donor DNA and is a variation of pBCSK+ in which a wild type HIV-1 donor DNA PCR product was inserted into pBCSK+ catalyzed by IN, resulting in the loss of two base pairs from the LTR ends. This plasmid was propagated in E. coli MC1061/P3 under the conditions described above. The integration acceptor was plasmid pBCSK+ (Stratagene, La Jolla, CA), which was propagated in E. coli DH5α. Plasmids were purified with Qiaprep columns (Qiagen, Chatsworth, CA) according to the manufacturer's instructions. The growth of DH5α containing pBCSK+ was selected for by addition of chloramphenicol (35 μg/ml). Integration donors were amplified by using thermostable Vent DNA polymerase and the primers listed above. Twenty-five pmol of each primer and 50 ng of pHHIV2 DNA, as the template, were used for each PCR reaction. Vent DNA polymerase was used according to the manufacturer's instructions. A total of 20 rounds of amplification were performed in each reaction. The amplification conditions were 94 °C for 2 min, 50 °C for 1 min, and 72 °C for 1 min for three rounds. This was followed by amplification conditions that used 94 °C for 2 min, 57 °C for 1 min, and 72 °C for 45 s for 17 additional rounds. The resultant product donor DNA was isolated after electrophoresis through 2% agarose gels equilibrated with 0.5× Tris borate-EDTA (3.Hindmarsh P. Ridky T. Reeves R. Andrake M. Skalka A.M. Leis J. J. Virol. 1999; 73: 2994-3003Crossref PubMed Google Scholar). The purified DNA (600 ng) was recovered using QIAquick gel extraction kit (Qiagen). The integration donors were ∼300 base pairs in length and were internally labeled during the PCR by the inclusion of [α-32P]dCTP (3000 Ci/mmol, 10 mCi/ml). The final concentrations of deoxyribonucleoside triphosphates during amplification reactions were 0.25 mm each of unlabeled dATP, dGTP, and dTTP. The final dCTP concentration was 0.0502 mm (12 Ci/mmol, 0.6 mCi/ml). The concerted integration reaction conditions were similar to those described by Hindmarsh et al. (3.Hindmarsh P. Ridky T. Reeves R. Andrake M. Skalka A.M. Leis J. J. Virol. 1999; 73: 2994-3003Crossref PubMed Google Scholar). Briefly, 15 ng (0.15 pmol of ends) of donor DNA was mixed with 50 ng of acceptor DNA (0.02 pmol) and 80 ng of HIV-1 IN (1.25 pmol) in a 8.5-μl preincubation reaction mixture containing, at final concentrations, 25 mm MOPS, pH 7.2, 23 mm NaCl, 10 mm dithiothreitol, 5% polyethylene glycol 8000, 10% dimethyl sulfoxide, 0.05% Nonidet P-40, 1% glycerol, 1.6 mm HEPES, pH 8.0, and 3.3 mmEDTA. The IN was diluted in a buffer containing 30% glycerol, 0.5m NaCl, 50 mm HEPES, pH 8.0, 1 mmdithiothreitol, and 0.1 mm EDTA. Where specified 100 ng of HMG-I(Y) was added to the reaction mixtures. The preincubation reaction mixtures were placed on ice overnight. The volume of each preincubation mixture was then increased to 10 μl with the addition of MgCl2 to a final concentration of 7.5 mm, and the integration assay mixture was incubated at 37 °C for 2 h. The reactions were stopped by increasing the volume to 150 μl by the addition of EDTA (final concentration of 4.25 mm), sodium dodecyl sulfate (final concentration of 0.44%), and proteinase K (final concentration of 0.06 mg/ml). After digestion for 60 min at 37 °C, the reaction mixtures were extracted with phenol followed by phenol-chloroform-isoamyl alcohol (25:24:1 mixture). Fifteen μl of 3m sodium acetate, pH 5.2, was added along with 1 μl of glycogen (10 mg/ml stock solution). The reaction products were precipitated by the addition of 450 μl of 100% ethanol and washed twice with 70% ethanol prior to electrophoresis and autoradiography. The reaction products were separated on a 1% agarose gel run in 0.5× Tris borate, EDTA, and ethidium bromide at 10 V/cm for 2 h. Following electrophoresis, gels were submerged in 5% trichloroacetic acid for 20 min or until the bromphenol blue dye turned bright yellow. After being washed with water, the gels were dried on DE-81 paper (Whatman) in a Bio-Rad slab gel dryer at 80 °C for ∼2 h under a vacuum. Quantitation of reaction products was carried out using a phosphorimaging device and ImageQuant 5.0 software. Experiments with wild-type donor integrants always accompanied experiments with mutant donor integrants as controls. All experiments were repeated at least two times. In all experiments, integration products were used directly for transformation of bacteria. The integration products were introduced into E. coliMCI061/P3 by electroporation, using a Bio-Rad electroporator with 0.1-cm electroporation cuvettes, 1.8-kV voltage, 25-μF capacitance, and 200-ohm resistance. The P3 episome is maintained at a low copy number. Therefore, only 40 μg/ml ampicillin, 15 μg/ml kanamycin, or 10 μg/ml tetracycline were required for selection. Under these conditions, we detected no colonies after supF selection when the donor, acceptor, or donor and acceptor were electroporated into cells in the absence of IN. Plasmid DNAs were recovered from individual clones, and integration junctions were sequenced by using primers U3seq (for sequencing the U3 junction) and U5seq (for sequencing the U5 junction). Sequencing was performed using the Thermo-Sequenase kit (U.S. Biochemical, Cleveland, Ohio). We used chi-square test to examine statistical significance of the difference between numbers of non-concerted events for different integration reactions. A binomial probability was used to determine significance of integration events into the same site in the target DNA. Since the total number of sequenced concerted integrants was 203 and the target plasmid length is 3400 base pairs, the formula used for calculations was the following:p = 203!/(x!(203-x)!)x(1–1/3400)(203-x), where x is a number of integration events into the same site. For calculation of probability of integration into a region we divided 3400 by the number of base pairs in the region. In the present study, we have used a reconstituted HIV-1 concerted DNA integration system that employs a specially designed mini donor DNA with HIV-1 U3 and U5 IN recognition sequences flanking a supF transcription unit (Fig. 1A), a supercoiled plasmid acceptor, purified bacterial-derived HIV-1 IN, and host HMG-I(Y) protein. The DNA integration products resulting from in vitro reactions have the characteristics associated with viral DNA integrated in vivo (3.Hindmarsh P. Ridky T. Reeves R. Andrake M. Skalka A.M. Leis J. J. Virol. 1999; 73: 2994-3003Crossref PubMed Google Scholar). When the products are analyzed by agarose gel electrophoresis, integration is detected by the insertion of the small donor into the larger acceptor DNA (see Fig. 1B). As much as 15% of the wild type donor DNA was converted to RFII products so that the molar amount of integrated donor DNA is half that of the target DNA in the reaction. The integration intermediates that are present in the different bands are diagrammatically represented to the right of the gel. RFIII products form via a nucleophilic attack of two one-ended integration events into the same site on the target plasmid. Thereby the amount of RFIII product is dependent on the donor DNA efficiency as an integrase substrate. If the U5 end, which is more active in one-ended events, is changed to be a less efficient substrate for integrase, the amount of RFIII product would decrease parallel to that of RFII product. Among the RFII-like products are integration intermediates that represent one-ended and two-ended donor insertions into the acceptor. The presence of one-ended versus two-ended donor insertions can be distinguished when the products of a reaction are introduced into bacteria (see Fig. 1C). One-ended donor integration products are not maintained and are thereby lost. Two-ended DNA integration products can be recovered from individual colonies and sequenced to establish the junctions between donor and target DNA. Examination of these junction sequences distinguishes whether the two-ended insertion products were derived by a concerted or a non-concerted mechanism. For instance, for a wild type donor, ∼93% of the two-ended insertion events have characteristics associated with a concerted DNA integration mechanism (Table I). This includes the loss of two base pairs from the ends of the LTRs, wide distribution of insertion sites of the donor into acceptor DNAs, and five base pair duplications introduced at the site of insertion. Only a small percentage of the two-ended integrants occurred by a non-concerted mechanism detected by deletions rather than five base pair duplications introduced into the acceptor (Table I).Table ISites of DNA integration of a wild type donor DNA into an acceptorDNA integration products from the HIV-1 reconstituted integration system were introduced into bacteria, and individual clones were isolated and sequenced as described under “Experimental Procedures.”a. Deoxyribonucleotide sequence of the junction of the donor integration into the acceptor DNA. The sequence for only the 3′-cleaved strand of the duplex for U5 and U3 is shown. Therefore the complementary strands of the duplex are presented. Lowercase letters denote duplication of the cell DNA; uppercase letters indicate the processed viral DNA sequences, which have lost two base pairs from each end unless otherwise indicated. Shaded entries are derived from a separate experiment than from non shaded entries. In a third experiment, the ratio of concerted to non-concerted integrants was 14:1.b. Zero denotes no base pair duplication indicative of a non-concerted DNA integration mechanism.c. Denotes deletion introduced into the acceptor DNA. Open table in a new tab DNA integration products from the HIV-1 reconstituted integration system were introduced into bacteria, and individual clones were isolated and sequenced as described under “Experimental Procedures.” a. Deoxyribonucleotide sequence of the junction of the donor integration into the acceptor DNA. The sequence for only the 3′-cleaved strand of the duplex for U5 and U3 is shown. Therefore the complementary strands of the duplex are presented. Lowercase letters denote duplication of the cell DNA; uppercase letters indicate the processed viral DNA sequences, which have lost two base pairs from each end unless otherwise indicated. Shaded entries are derived from a separate experiment than from non shaded entries. In a third experiment, the ratio of concerted to non-concerted integrants was 14:1. b. Zero denotes no base pair duplication indicative of a non-concerted DNA integration mechanism. c. Denotes deletion introduced into the acceptor DNA. We reexamined the effect of removing one of the two IN recognition sequences from the ends of the HIV-1 donor DNA. When the U5 IN recognition sequence was replaced by random sequence, the RFII-like product detected by gel electrophoresis decreased 89% compared with wild type (Fig. 2A, lanes 1 and 2). When the U3 IN recognition sequence was replaced by random sequence, the RFII-like product decreased only 10% compared with wild type (Fig. 3A, lane 3). As a control, when both IN recognition sequences were deleted, no integration into the acceptor DNA was detected on the gels (Fig. 3A, lane 2). When the products from the ΔU3 or ΔU5 reaction were introduced into bacteria, no colonies above the background level were obtained indicating that the removal of either of these IN recognition sequences resulted in the loss of two-ended DNA integration products (data not shown). The finding that there was very little decrease in the RFII-like products observed with the ΔU3 donor DNA indicates that the U5 IN recognition sequence efficiently promotes one-ended insertion events in the absence of a U3 IN recognition sequence.Figure 3Effect of substitutions in the HIV-1 U3 IN recognition sequence on integration in vitro. A, gel electrophoresis analysis of integration products from reactions with wild type donor (lane 1), donor with both IN recognition sequences substituted by random sequences (lane 2), donor with the U3 LTR end substituted by random sequence (lane 3), or donors with wild type U5 LTR end (lanes 3, 4, 5, 6) or deleted U5 LTR end (replaced by random sequence) (lanes 7, 8, 9) and containing inversion mutations in U3 at positions 5–6 (lanes 4, 7), positions 4–7 (lanes 5, 8), and positions 3–4 (lanes 6, 9). B, quantification of RFII products shown in A (closed bars) and total number of colonies containing two-ended integrants (open bars) as described in the legend to Fig. 2. The data shown is an average of two independent experiments, the standard deviation between experiments was 1–2%. C, percent of integrants derived from Table V, Table VI, Table VII formed by a concerted mechanism involving two ends of the same donor DNA.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The effect on reconstituted integration of base pair inversions into positions 5–6 (TAGCAGT → TTCCAGT) and 4–7 (TAGCAGT → ATCGAGT) of an HIV-1 mini donor DNA is presented in Fig. 2. We also analyzed the effect of substitution of the conserved CA dinucleotide at positions 3–4 (TAGCAGT → TAGGGGT). Integration reactions were carried out as described under “Experimental Procedures”, and products were analyzed first by agarose gel electrophoresis. Base pair substitutions introduced at positions 5–6 into the U5 LTR IN recognition sequence caused more than a 2-fold decrease of integration efficiency compared with wild type (Fig. 2A, lane 3). Substitutions at positions 4–7 and 3–4 resulted in more dramatic decreases of integration efficiency (Fig. 2A, lanes 4 and 5). Biological selection of integration products after their introduction in bacteria showed that when substitutions were made at positions 5–6 the number of two-ended integrants (judged by the number of recovered colonies) decreased to 40% that of a wild type donor (Fig. 2B). The number of selected double-ended integrants that resulted from reactions with donors containing substitutions at positions 4–7 and 3–4 was 5.7 and 3%, respectively, that of wild type (Fig. 2B). These decreases followed the percentage loss of integrants detected by the gel electrophoresis analysis (Fig. 2B). We further analyzed the products recovered from bacteria by sequencing individual integrants. In comparison to wild type, a donor with base pair inversions at positions 5–6 caused a 3-fold increase in non-concerted integration events, which introduced deletions rather than small duplications into the acceptor DNA (Table II). This is a statistically significant result as the probability of its happening is p = 0.04. Interestingly, the larger base pair inversions at positions 4–7, which includes positions 5–6, resembled wild type in that most integrants arose by a concerted mechanism (Fig. 2C, Table III).Table IISites of DNA integration of a HIV-1 donor DNA with base pair substitutions at U5–5GA6 positionsDNA integration products from the HIV-1 reconstituted integration system were introduced into bacteria, and individual clones were isolated and sequenced as described under “Experimental Procedures.”a. Deoxyribonucleotide sequence of the junction of the donor integration into the acceptor DNA. All notations are as in the legend to Table I. Shaded entries are from a separate experiment than from non shaded entries. In a third experiment, the ratio of concerted to non concerted integrants was 9:3.b. Zero denotes no base pair duplication indicative of a non-concerted DNA integration mechanism.c. Denotes deletion introduced into the acceptor DNA.d. Denotes a 19-base pair deletion in the donor DNA in the U5 LTR end that uses the first internal CA dinucleotide. Open table in a new tab Table IIISites of DNA integration of a HIV-1 donor DNA with base pair substitutions at U5–4CGAT7 positionsDNA integration products from the HIV-1 reconstituted integration system were introduced into bacteria, and individual clones were isolated and sequenced as described under “Experimental Procedures.”a. Deoxyribonucleotide sequence of the junction of" @default.
• W1978523876 created "2016-06-24" @default.
• W1978523876 creator A5014262516 @default.
• W1978523876 creator A5017138813 @default.
• W1978523876 date "2002-03-01" @default.
• W1978523876 modified "2023-10-17" @default.
• W1978523876 title "Changes in the Mechanism of DNA Integration in Vitro Induced by Base Substitutions in the HIV-1 U5 and U3 Terminal Sequences" @default.
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