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• W1994576403 abstract "The U3 and U5 termini of linear retrovirus DNA contain imperfect inverted repeats that are necessary for the concerted insertion of the termini into the host chromosome by viral integrase. Avian myeloblastosis virus integrase can efficiently insert the termini of retrovirus-like DNA donor substrates (480 base pairs) by a concerted mechanism (full-site reaction) into circular target DNA in vitro. The specific activities of virion-derived avian myeloblastosis virus integrase and bacterial recombinant Rous sarcoma virus (Prague A strain) integrase (∼50 nm or less) appear similar upon catalyzing the full-site reaction with 3′-OH recessed wild type or mutant donor substrates. We examined the role of the three nonsymmetrical nucleotides located at the 5th, 8th, and 12th positions in the U3 and U5 15-base pair inverted repeats for their ability to modify the full-site and simultaneously, the half-site strand transfer reactions. Our data suggest that the nucleotide at the 5th position appears to be responsible for the 3–5-fold preference for wild type U3 ends over wild type U5 ends by integrase for concerted integration. Additional mutations at the 5th or 6th position, or both, of U3 or U5 termini significantly increased (∼3 fold) the full-site reactions of mutant donors over wild type donors. The U3 and U5 termini of linear retrovirus DNA contain imperfect inverted repeats that are necessary for the concerted insertion of the termini into the host chromosome by viral integrase. Avian myeloblastosis virus integrase can efficiently insert the termini of retrovirus-like DNA donor substrates (480 base pairs) by a concerted mechanism (full-site reaction) into circular target DNA in vitro. The specific activities of virion-derived avian myeloblastosis virus integrase and bacterial recombinant Rous sarcoma virus (Prague A strain) integrase (∼50 nm or less) appear similar upon catalyzing the full-site reaction with 3′-OH recessed wild type or mutant donor substrates. We examined the role of the three nonsymmetrical nucleotides located at the 5th, 8th, and 12th positions in the U3 and U5 15-base pair inverted repeats for their ability to modify the full-site and simultaneously, the half-site strand transfer reactions. Our data suggest that the nucleotide at the 5th position appears to be responsible for the 3–5-fold preference for wild type U3 ends over wild type U5 ends by integrase for concerted integration. Additional mutations at the 5th or 6th position, or both, of U3 or U5 termini significantly increased (∼3 fold) the full-site reactions of mutant donors over wild type donors. Upon retrovirus infection, the viral RNA genome is reverse transcribed into a linear blunt ended DNA genome. The retrovirus U3 and U5 DNA termini contain LTR 1The abbreviations used are: LTR, long terminal repeat; AMV, avian myeloblastosis virus; bp, base pair(s); RSV, Rous sarcoma virus; PrA, Prague A strain; wt, wild type; kbp, kilobase pair(s). sequences with short imperfect inverted repeats located at the very end of the blunt ended LTRs (1Varmus H.E. Shapiro J.A. Mobile Genetic Elements. Academic Press, New York1983: 411-503Google Scholar). In vivo, the inverted repeats are necessary for virally encoded integrase to catalyze the removal of a dinucleotide from the 3′-OH termini and the subsequent full-site integration reaction (2Goff S.P. Annu. Rev. Genet. 1992; 26: 527-544Crossref PubMed Scopus (212) Google Scholar). The full-site reaction involves the concerted insertion of the two recessed LTR DNA termini into the host genome. This reaction also results in the formation of a small size host duplication at the site of insertion whose size is virus-specific (1Varmus H.E. Shapiro J.A. Mobile Genetic Elements. Academic Press, New York1983: 411-503Google Scholar). In vitro, the mechanisms involved in the recognition of the blunt ended LTR termini by integrase for the 3′-OH processing reaction and for half-site strand transfer of the recessed LTR termini into target DNA have been investigated (2Goff S.P. Annu. Rev. Genet. 1992; 26: 527-544Crossref PubMed Scopus (212) Google Scholar, 3Bushman F.D. Craigie R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1339-1343Crossref PubMed Scopus (355) Google Scholar, 4Bushman F.D. Fujiwara T. Craigie R. Science. 1990; 249: 1555-1558Crossref PubMed Scopus (266) Google Scholar, 5Ellison V. Brown P.O. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7316-7320Crossref PubMed Scopus (152) Google Scholar, 6Katz R.A. Merkel G. Kulkosky J. Leis J. Skalka A. Cell. 1990; 63: 87-95Abstract Full Text PDF PubMed Scopus (297) Google Scholar, 7Lee S.P. Kim H.G. Censullo M.L. Han M.K. Biochemistry. 1995; 34: 10205-10214Crossref PubMed Scopus (45) Google Scholar, 8Reicin A.S. Kalpana G. Paik S. Marmon S. Goff S. J. Virol. 1995; 69: 5904-5907Crossref PubMed Google Scholar, 9Van Den Ent F.M.I. Vink C. Plasterk R.H.A. J. Virol. 1994; 68: 7825-7832Crossref PubMed Google Scholar, 10Vink C. van der Linden K.H. Plasterk R.H.A. J. Virol. 1994; 68: 1468-1474Crossref PubMed Google Scholar). The half-site reaction involves the insertion of only one LTR terminus into the DNA target. These in vitro analyses using purified integrase from several retrovirus species have established that the imperfect inverted repeat sequences located at the LTR termini are also necessary for catalysis (1Varmus H.E. Shapiro J.A. Mobile Genetic Elements. Academic Press, New York1983: 411-503Google Scholar, 11Katz R.A. Skalka A. Annu. Rev. Biochem. 1994; 63: 133-163Crossref PubMed Scopus (535) Google Scholar, 12Vink C. Plasterk R.H.A. Trends Genet. 1993; 9: 4333-4348Abstract Full Text PDF Scopus (104) Google Scholar). Besides the essential CA dinucleotide located 2 nucleotides downstream from the blunt ended viral termini, approximately 5–10 nucleotides internally also play varying roles in the 3′-OH processing and half-site strand transfer reactions (2Goff S.P. Annu. Rev. Genet. 1992; 26: 527-544Crossref PubMed Scopus (212) Google Scholar,13Balakrishnan M. Jonsson C.B. J. Virol. 1997; 71: 1025-1035Crossref PubMed Google Scholar). The full-site integration reaction can be catalyzed efficiently using retrovirus-like donor substrates with integrase purified from virions (14Vora A.C. Grandgenett D.P. J. Virol. 1995; 69: 7483-7488Crossref PubMed Google Scholar, 15Vora 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). Study of the specific interactions of integrase with the U3 and U5 LTR termini for the full-site integration reaction in vitro would provide insights into the in vivointegration reaction. The bimolecular donor full-site reaction (see Fig. 1, bottom) catalyzed by AMV integrase produces the avian 6-bp host site duplication as demonstrated by DNA sequence analysis (14, 15, this report). The unimolecular donor full-site reaction, where two ends of one molecule are used, is less than 5% as efficient as the bimolecular reaction (15Vora 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, 16Aiyar A. Hindmarsh P. Skalka A. Leis L. J. Virol. 1996; 70: 3571-3580Crossref PubMed Google Scholar, 17Fitzgerald M.L. Grandgenett D.P. J. Virol. 1994; 68: 4314-4321Crossref PubMed Google Scholar, 18Fitzgerald M.L. Vora A.C. Zeh W.G. Grandgenett D.P. J. Virol. 1992; 66: 6257-6263Crossref PubMed Google Scholar). The ability of bacterial recombinant RSV integrase (16Aiyar A. Hindmarsh P. Skalka A. Leis L. J. Virol. 1996; 70: 3571-3580Crossref PubMed Google Scholar) and AMV integrase (17Fitzgerald M.L. Grandgenett D.P. J. Virol. 1994; 68: 4314-4321Crossref PubMed Google Scholar, 18Fitzgerald M.L. Vora A.C. Zeh W.G. Grandgenett D.P. J. Virol. 1992; 66: 6257-6263Crossref PubMed Google Scholar) to produce the 6-bp host duplication with the unimolecular donor reaction is also high. We have reported previously that the avian retrovirus U3 LTR terminus is preferred over the U5 LTR terminus for both the 3′-OH processing (19Fitzgerald M.L. Vora A.C. Grandgenett D.P. Anal. Biochem. 1991; 196: 19-23Crossref PubMed Scopus (14) Google Scholar) and the half-site and full-site strand transfer reactions (14Vora A.C. Grandgenett D.P. J. Virol. 1995; 69: 7483-7488Crossref PubMed Google Scholar, 15Vora 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,18Fitzgerald M.L. Vora A.C. Zeh W.G. Grandgenett D.P. J. Virol. 1992; 66: 6257-6263Crossref PubMed Google Scholar, 19Fitzgerald M.L. Vora A.C. Grandgenett D.P. Anal. Biochem. 1991; 196: 19-23Crossref PubMed Scopus (14) Google Scholar, 20Grandgenett D.P. Inman R.B. Vora A.C. Fitzgerald M.L. J. Virol. 1993; 67: 1628-1636Crossref Google Scholar). Three nonsymmetrical nucleotides are at the 5th, 8th, and 12th positions of the 15-bp inverted repeats located at the RSV LTR termini. It is unknown what role the three nonsymmetrical nucleotides have in modifying the recognition of integrase for either LTR terminus or their influence on the full-site integration reaction. In this report we examined the role of the three nonsymmetrical nucleotides located in the avian U3 and U5 15-bp inverted repeat and several other LTR mutations in the formation of donor-target recombinants that are produced by the full-site integration reaction. The full-site reactions were catalyzed by either AMV integrase derived from virions or by recombinant RSV PrA integrase purified from bacteria. Of the seven LTR mutations examined, some had either a significant gain or a loss of function for the full-site reaction, whereas others had no affect. The interactions of integrase with wt and mutant LTR DNA termini for full-site strand transfer were evaluated byBglII digestion of labeled donor-target recombinants resolved by agarose gel electrophoresis and DNA sequence analysis of donor-target junctions. The specific activities of both virion and recombinant integrase appear equivalent when catalyzing the half-site and full-site strand transfer reactions with either wt or mutant LTR DNA substrates, or the 3′-OH processing reaction with similar blunt ended substrates. The wt and mutant LTR donor substrates were produced by cloning 60-bp double-stranded oligonucleotides containing terminal U3 and U5 LTR sequences into the NdeI site of pUC19 plasmids lacking a HindIII site (Fig. 1,top). The U3 and U5 ends were separated by aHindIII site. A genetic selection marker, thesupF gene (420 bp), was cloned into the HindIII site of each plasmid. The clones were sequenced to verify the constructs. All plasmids were purified by velocity sedimentation before digestion with either NdeI or both NdeI andHinfI to isolate the appropriate LTR donor fragments. The donor fragments were isolated by either agarose or by polyacrylamide gel electrophoresis. All donor substrates contained the uniqueBglII site adjacent to the U3 LTR terminus (Fig. 1,bottom). With this same structural arrangement, all of the donor-target recombinants would give the same restriction pattern. The target DNA was supercoiled pGEM, which lacked a BglII site. The 5′-end labeling ofNdeI-digested DNA fragments was performed by T4 polynucleotide kinase and [γ-32P]ATP (15Vora 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). We routinely labeled sets of wt and mutant donor substrates at the same time to produce labeled molecules that had very similar specific activities, usually at 2,000 cpm/ng of DNA. This labeling strategy allowed us to determine directly by PhosphorImager analysis the amount of input donor DNA which was used for strand transfer within each set of wt and mutant donor reactions. The 3′-OH labeling of the NdeI sites on the 480-bp donors was accomplished by using Klenow polymerase with [α-32P]TTP and unlabeled deoxynucleotides (19Fitzgerald M.L. Vora A.C. Grandgenett D.P. Anal. Biochem. 1991; 196: 19-23Crossref PubMed Scopus (14) Google Scholar). The fill-in reactions were always greater than 98% as verified by restriction enzyme cleavage of the labeled molecules and by subsequent examination of the labeled strand on DNA sequencing gels. After labeling, the donors were digested with HinfI producing two fragments of 248 bp (U5 end) and 198 bp (U3 end) in size. TheHinfI sites were filled in with unlabeled deoxynucleotides. Scale-up reactions (40×) were performed for sequencing of donor-target recombinants. After isolation of the 3.3-kbp donor-target recombinants by BglII restriction enzyme digestion and agarose gel electrophoresis, the ligated DNAs were transformed into CA244 cells (15Vora 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). Colonies were screened for plasmids that were analyzed by size, restriction enzyme digestion, and dideoxy DNA sequencing to determine the size of the host site duplications. Standard reaction mixtures (20 μl) for full-site strand transfer contained 20 mm HEPES buffer (pH 7.5), 1 mm dithiothreitol, 8% polyethylene glycol 6000, 15% dioxane, and 330 mm NaCl (14Vora A.C. Grandgenett D.P. J. Virol. 1995; 69: 7483-7488Crossref PubMed Google Scholar). Briefly, AMV or RSV integrase (50 nm) was first preincubated on ice for 10 min in the above assay mixture with 15 ng of donor DNA. The strand transfer reactions were initiated by the addition of target (100 ng) and immediate incubation at 37 °C for 10 min. The molar ratio of dimeric integrase to donor ends was 12:1, respectively. The reactions were stopped, and the DNA products were separated on agarose gels (15Vora 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). The amount of products produced was determined by a Molecular Dynamics PhosphorImager. The standard 3′-OH processing reaction conditions were 10 mm HEPES (pH 7.5), 2 mm dithiothreitol, 140 mm NaCl, and 20 mm MgCl2 (19Fitzgerald M.L. Vora A.C. Grandgenett D.P. Anal. Biochem. 1991; 196: 19-23Crossref PubMed Scopus (14) Google Scholar). AMV and RSV integrase was at either 10 or 20 nm. Integrase:blunt ended donor substrates ratios used for the 3′-OH processing reaction were similar to those described for strand transfer. AMV (21Grandgenett D.P. Vora A.C. Schiff R. Virology. 1978; 89: 119-132Crossref PubMed Scopus (98) Google Scholar) and RSV integrase (data not shown) were purified to near homogeneity. RSV integrase was cloned from an infectious PrA viral DNA clone (22Mumm S.R. Grandgenett D.P. J. Virol. 1991; 65: 1160-1167Crossref PubMed Google Scholar) and expressed in bacteria using a pET11 vector. The purification procedure and the physical characterization of RSV integrase will be published separately. The protein concentrations of each purified integrase preparation were determined by A 280 measurements. In this report we examined the contribution of the three nonsymmetrical nucleotides that map to the 5th, 8th, and 12th positions of the RSV U3 and U5 LTR DNA termini for the full-site integration reaction (Fig. 1). The mutations were introduced onto the 3′-OH processing strand and are numbered with respect to the blunt ended terminus. The U3 nonsymmetrical nucleotides were modified singly to U5 sequences, and the U5 nonsymmetrical nucleotides were modified singly to U3 sequences; in each case, the other two nonsymmetrical nucleotides were unchanged. Several other gain or loss of function mutations in the LTR termini were also examined. In all cases, a wt LTR terminus was present on the opposite end of each LTR mutant donor substrate. The full-site and half-site strand transfer reactions using the 480-bp wt U3/U5, wt U3/U3, and three mutant LTR donor substrates (Fig. 1) with AMV integrase at 50 nm are shown in Fig. 2 A. The wt U3/U5 and wt U3/U3 donors served as control substrates for measuring catalytic rates and for determining how the different wt LTR termini affect the full-site reaction. In a 10-min reaction at 37 °C, the total incorporation of each input donor into the pGEM target was 10.8, 15, 3.7, 10.8, and 16.3% for wt U3/U5, wt U3/U3, U3P5-A/T, U3P8-A/G, and U5P5,6-TT/AA, respectively (Fig. 3,bottom line). As determined by PhosphorImager analysis, the above reactions were linear for at least 20 min, and in most cases, half or more of the DNA products were full-site recombinants. With the same labeled LTR donors, purified RSV integrase at 50 nmincorporated 9.4, 14.3, 1.8, 8, and 22% of the input substrates, respectively, into the pGEM target (Fig. 2 B). With either integrase, fewer than 1% of the input donors were integrated into themselves as donor-donor recombinants. This is the first report of a recombinant integrase protein that has a specific activity similar to that of the virion-purified protein for catalyzing the full-site reaction using wt and mutant donor substrates. It should be noted that concentrations of AMV integrase greater than ∼125 nm (14Vora A.C. Grandgenett D.P. J. Virol. 1995; 69: 7483-7488Crossref PubMed Google Scholar) or RSV integrase greater than ∼60 nm (data not shown) in the standard reaction for full-site integration initiate inhibition of catalysis.Figure 3Quantitative analysis of BglII restriction products of donor-target recombinants produced by AMV integrase. For easier visualization and understanding of the tabulated data in this figure, please see the BglII digestion data in Fig. 4 A. The total amounts of donor-target recombinants produced by AMV integrase with wt U3/U5, wt U3/U3, U3P5-A/T, U3P8-A/G, and U5P5,6-TT/AA donors (Fig. 2 A) are shown on the bottom line as a percent of donor substrate inserted into pGEM. The vertical columns in thebox were identified with each of the above donors. Schematics for the BglII digestion products are shown in theleft margin. The BglII digestion of one donor molecule inserted into pGEM produces either “A” or “B” type structures depending on whether the U5 or U3 end was integrated, respectively. BglII digestion of the linear 3.8-kbp donor-target recombinant produces four linear restriction fragments with the two 3.3-kbp U3/U5 fragments comigrating. Using PhosphorImager data, the percentage of each digested product produced was determined relative to the total donor-target products produced by that donor (Fig. 4 A). The BglII 3.7-kbp U5/U5 product (middle of first vertical column of numbers) obtained using wt U3/U5 donor was set to equal 1. The other numbers were derived by dividing the percentage of homologous U5/U5 full-site product of the wt donor into the percentage of each product obtained with a specific donor. For example, the numbers in the first column (wt U3/U5 donor) are very similar to the numbers in thefourth column (U3P8-A/G donor) because this mutation did not modify significantly the half-site and full-site reactions catalyzed by integrase. For visualization of the above comparison, comparelanes 2 and 8 of Fig. 4 A.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Modification of the 5th nucleotide (A to T) on the U3 LTR terminus (U3P5-A/T donor) decreased the ability of both AMV integrase and RSV integrase to catalyze the full-site and half-site reactions to one-third or less of that obtained with the wt U3/U5 donor (Fig. 2,A and B, lanes 2 and 6; Fig. 3). Modification of the nonsymmetrical 8th nucleotide of U3 from A to G (U3P8-A/G donor) did not modify the ability of either integrase to catalyze stand transfer compared with the wt U3/U5 donor (Fig. 2,A and B, lanes 2 and 8). A gain of function mutation in the U5 LTR, the U5P5,6-TT/AA donor relative to the wt U3/U5 donor, is shown in Fig. 2, A andB, lanes 2 and 10; and Fig. 3. The double mutation produces a U3 LTR sequence up to the 5th nucleotide with one additional nucleotide change upstream. Analysis of the data shows that the 5th and 6th nucleotides that are critical for half-site strand transfer also significantly influence the full-site reactionin vitro. We investigated if the individual mutations that were introduced into the recessed U3 and U5 LTR termini modified their interactions with other wt LTR termini or with themselves for full-site strand transfer and, simultaneously, for the half-site reaction. To investigate these interactions, the wt and mutant donor-target recombinants (Fig. 2, A andB) were subjected to BglII restriction analysis (AMV reactions, Fig. 4 A; RSV reactions, Fig. 4 B). It was established previously thatBglII digestion of half-site and full-site donor-target recombinants gives specific cleavage patterns (14Vora A.C. Grandgenett D.P. J. Virol. 1995; 69: 7483-7488Crossref PubMed Google Scholar, 15Vora 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), as illustrated in Figs. 1 and 3. The quantity of each uncleaved and cleaved donor-target recombinant was determined by PhosphorImager analysis. For comparison among all donor-target recombinants, the full-site 3.7-kbp homologous U5/U5 recombinant obtained by BglII digestion of the wt U3/U5 donor (Fig. 4 A, lane 2) for AMV integrase was arbitrarily set to 1 (Fig. 3), in relationship to the other digested donor-target recombinants obtained with the other donor substrates. The full-site U5/U5 recombinants were produced at the lowest level for any of the wt full-site recombinants. Similar quantitative data (data not shown) were obtained as shown in Fig. 3with the RSV integrase reactions with the same donor substrates (Fig.4 B). A control strand transfer reaction was analyzed first with the wt U3/U5 donor substrate (Fig. 4, A and B, lanes 1 and 2; Fig. 3). With this donor, the recessed U3 LTR terminus was approximately 3-fold more effective than the recessed U5 LTR terminus for the half-site reaction (see column of wt U3/U5 donor in Fig. 3 and the column of BglII-digested products in Fig.4, A or B, lane 2). TheBglII-digested half-site donor-target recombinants were classified as “A” and “B” type structures that were produced by the use of either the U5 or U3 LTR terminus, respectively (Fig. 3). For the full-site reactions, the 2.9-kbp homologous U3/U3 recombinants were ∼5-fold higher than the 3.7-kbp homologous U5/U5 recombinants whose quantity was set to 1. The full-site 3.3-kbp U3/U5 recombinants were ∼3.6-fold higher than the U5/U5 recombinants. The data suggest that integrase recognizes and subsequently uses the U3 terminus at a significantly higher level than the U5 LTR terminus when the LTRs are presented at an equal molar ratio in the reaction mixture. The BglII digestion of control wt U3/U3 donor reactions (Fig. 3, second column; Fig. 4, A andB, lane 4) demonstrated that the “A” and “B” half-site reactions were nearly equal, suggesting that sequences outside the 25-bp LTR region appeared not to influence strand transfer significantly. All full-site reactions with this donor would be homologous U3/U3 reactions only. The full-site 3.7-kbp and 2.9-kbp U3/U3 recombinants were nearly equal but were half the amount of the full-site 3.3-kbp U3/U3 recombinants. The individual wt U3/U3 donors can be inserted into pGEM in two orientations giving rise to the same size 3.3-kbp BglII restriction fragment. We cannot rule out the possibility of a minor negative effect exerted by the proximity ofsupF sequences located at the BglII side of the wt U3/U3 donor. The effect is seen when comparing the “B” with “A” half-site products or the full-site 2.9 kbp with 3.7 U3/U3 recombinants (Fig. 3, second column). The two control reactions above demonstrate that BglII restriction analysis of the donor-target recombinants provides a quantitative and convenient method of simultaneously analyzing half-site and full-site integration reactions. Because the U3 terminus is identical to the U5 terminus in sequence except for the nonsymmetrical nucleotides, the ability of integrase to recognize and to use recessed U3 over U5 LTR ends would be related to these nucleotides. We next examined how the individual nucleotide changes in the U3 LTR terminus modified the ability of integrase to catalyze both half-site reactions and full-site reactions with its own mutated U3 terminus and with wt U5 LTR ends. With the mutant U3P5-A/T donor and AMV integrase at 50 nm (Fig. 4 A, lanes 5 and 6), the overall strand transfer products were decreased to one-third the level observed with the wt U3/U5 donor (Fig.3). Increasing the concentration of AMV integrase to 88 or 120 nm did not increase the efficiency of catalysis of donor U3P5-A/T nor change its BglII restriction pattern relative to the wt U3/U5 donor (data not shown). The BglII digestion pattern observed with the mutant donor U3P5-A/T was changed significantly relative to the wt U3/U5 donor (Fig.4 A, lanes 2 and 6). The most striking observation is that the U3P5-A/T LTR end appears to have activity similar to the wt U5 LTR end located on the same donor molecule. Almost all of the catalytic reactions with the U3P5-A/T donor more closely parallel wt U5 LTR than wt U3 LTR activities (Fig. 3, firstand third columns). In addition, the half-site B U3P5-A/T LTR reaction of donor U3P5-A/T was inhibited 60% compared with the U3 LTR of wt donor U3/U5. The full-site 3.3-kbp U3/U5 and the 2.9-kbp homologous U3/U3 recombinant reactions of donor U3P5-A/T were decreased 50 and 85%, respectively, compared with the same wt U3/U5 donor reactions. Similar results were obtained with the BglII digestion patterns using donor U3P5-A/T with RSV integrase at 50 nm (Fig. 4 B, lane 6). With either AMV or RSV integrase (Fig. 4, A and B, lanes 2 and 8), changing the 8th nucleotide of the U3 LTR from A to G (donor U3P8-A/G) (Fig. 3, fourth column) had no apparent affect on the strand transfer reactions compared with the wt U3/U5 donor. The 12th nonsymmetrical nucleotide in the U3 inverted repeat was not examined, although a single nucleotide deletion 2 bp downstream of the 8th nucleotide decreases half-site and full-site catalytic rates (see below). The data suggest that the nonsymmetrical nucleotide at the 5th position has a significant effect on the preferential recognition of U3 ends over U5 ends by integrase as well as on subsequent catalysis. The wt U5 end of donor U3P5-A/T was able to relieve some of the inhibitory effects of the single nucleotide change in the U3P5-A/T terminus. This conclusion was reached if one compares the ratio of the full-site U3/U5 (3.3 kbp) to homologous U3/U3 (2.9 kbp) reactions observed (0.66) with the wt U3/U5 donor (Fig. 3, first column) to the ratio observed (2.2) with the full-site 3.3-kbp to 2.9-kbp reactions of donor U3P5-A/T (Fig. 3, third column). We next examined the effects of three U5 mutations individually directed against the nonsymmetrical nucleotides on the strand transfer reactions. We compared the wt U3/U5 donor with donors containing single mutations in U5 at the 5th, 8th, and 12th positions (U5P5-T/A, U5P8-G/A, U5P12-G/T, respectively) using both AMV integrase (Fig.5 A) and RSV integrase (Fig.5 B). To determine if sequences downstream of the 8th nonsymmetrical nucleotide of U3 were important, we also investigated whether a single nucleotide deletion (A at position 10) in the U3 LTR of donor U5P8-G/A had an effect on catalysis. For a 10-min reaction at 37 °C, AMV and RSV integrase at 50 nm incorporated 10.6, 12.6, 6, and 12% and 10.6, 12.8, 8.4, and 14% of the above donors into pGEM, respectively. As shown previously, fewer than 1% of the donors were integrated into themselves, and greater than 50% of the donor-target recombinants were full-site products (Fig. 5 and data not shown). The BglII digestion fragments observed in Fig. 5 with the U5 LTR mutations were subjected to PhosphorImager analysis as described in Fig. 3 (data not shown). The data demonstrated that the wt U3/U5 donor reactions with AMV integrase (Fig. 5 A, lanes 1and 2) and RSV integrase (Fig. 5 B, lanes 1 and 2) were essentially identical to those observed in Fig. 4 and as calculated in Fig. 3. Several changes in the half-site and full-site reactions were evident with the above set of U5 LTR mutations. As expected, the modification of the U5 LTR sequence to a U3 LTR sequence (T to A at the 5th position; donor U5P5-T/A) up-regulated the ability of both integrase proteins to use the mutated U5 terminus, which is now similar to the wt U3 terminus. For example, the half-site (“A”) product and the full-site 3.7-kbp recombinants using the mutated U5 terminus of donor U5P5-T/A (Fig. 5, A orB, lane 4) were significantly higher (2–3-fold) than the same size products using the U5 end of the wt U3/U5 donor, shown in lane 2 of panels A and B. Similarly, the half-site wt U3 product (“B”) of donor U5P5-T/A is nearly the same quantity as the half-site product (“A”) with the mutated U5 terminus of the same donor (Fig. 5, A andB, lane 4). In lane 4 of both integrase sets, the mutated U5 end interactions with the wt U3 end to produce the full-site 3.3-kbp recombinants were similar in quantity to the full-site 2.9-kbp homologous U3/U3 product obtained with the wt U3/U5 donor shown in lane 2. The changing of the 8th (donor U5P8-G/A) or 12th (donor U5P12-G/T) position of the U5 LTR to U3 sequences (Fig. 5, A and B, lanes 6and 8, respectively) had little effect on the mutated U5 LTR strand transfer reactions compared with reactions observed with the U5 LTR of the wt U3/U5 donor (lane 2). The modification of the U3 LTR by a single nucleotide deletion at the 10th position of donor U5P8-G/A had a modest effect on the overall catalytic rates (comparelanes 5 and 6 of 5A and 5Bwith t" @default.
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