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Presteady-state Analysis of Avian Sarcoma Virus Integrase

2002; Elsevier BV; Volume: 277; Issue: 14 Linguagem: Inglês

10.1074/jbc.m111314200

1083-351X

Kogan K. Bao, Anna Marie Skalka, Isaac Wong,

HIV Research and Treatment

The integrase-catalyzed insertion of the retroviral genome into the host chromosome involves two reactions in vivo: 1) the binding and endonucleolytic removal of the terminal dinucleotides of the viral DNA termini and 2) the recombination of the ends with the host DNA. Kukolj and Skalka (Kukolj, G., and Skalka, A. M. (1995) Genes Dev. 9, 2556–2567) have previously shown that tethering of the termini enhances the endonucleolytic activities of integrase. We have used 5′-5′ phosphoramidites to design reverse-polarity tethers that allowed us to examine the reactivity of two viral long terminal repeat-derived sequences when concurrently bound to integrase and, additionally, developed presteady-state assays to analyze the initial exponential phase of the reaction, which is a measure of the amount of productive nucleoprotein complexes formed during preincubation of integrase and DNA. Furthermore, the reverse-polarity tether circumvents the integrase-catalyzed splicing reaction (Bao, K., Skalka, A. M., and Wong, I. (2002) J. Biol. Chem. 277, 12089–12098) that obscures accurate analysis of the reactivities of synapsed DNA substrates. Consequently, we were able to establish a lower limit of 0.2 s−1 for the rate constant of the processing reaction. The analysis showed the physiologically relevant U3/U5 pair of viral ends to be the preferred substrate for integrase with the U3/U3 combination favored over the U5/U5 pair. The integrase-catalyzed insertion of the retroviral genome into the host chromosome involves two reactions in vivo: 1) the binding and endonucleolytic removal of the terminal dinucleotides of the viral DNA termini and 2) the recombination of the ends with the host DNA. Kukolj and Skalka (Kukolj, G., and Skalka, A. M. (1995) Genes Dev. 9, 2556–2567) have previously shown that tethering of the termini enhances the endonucleolytic activities of integrase. We have used 5′-5′ phosphoramidites to design reverse-polarity tethers that allowed us to examine the reactivity of two viral long terminal repeat-derived sequences when concurrently bound to integrase and, additionally, developed presteady-state assays to analyze the initial exponential phase of the reaction, which is a measure of the amount of productive nucleoprotein complexes formed during preincubation of integrase and DNA. Furthermore, the reverse-polarity tether circumvents the integrase-catalyzed splicing reaction (Bao, K., Skalka, A. M., and Wong, I. (2002) J. Biol. Chem. 277, 12089–12098) that obscures accurate analysis of the reactivities of synapsed DNA substrates. Consequently, we were able to establish a lower limit of 0.2 s−1 for the rate constant of the processing reaction. The analysis showed the physiologically relevant U3/U5 pair of viral ends to be the preferred substrate for integrase with the U3/U3 combination favored over the U5/U5 pair. The insertion of a DNA copy of the viral RNA genome into the host chromosome is a critical step in the reproduction cycle of retroviruses (1.Katz R.A. Skalka A.M. Annu. Rev. Biochem. 1994; 63: 133-173Crossref PubMed Scopus (535) Google Scholar). Integrase catalyzes this reaction via a 2-step process, 1) the processing reaction, which is the recognition and endonucleolytic "trimming" of DNA sequences at the two 3′-ends of linear viral DNA and 2) the joining reaction, which is the concerted cleavage-ligation of the processed ends into the host chromosomal DNA. In avian sarcoma virus (ASV), 1The abbreviations used are: ASVavian sarcoma virusINintegraseLTRlong terminal repeatA260absorbance at 260 nmTBETris borate-EDTA the processing reaction produces site-specific cuts at the CATT sequence of the viral 3′-ends, removing the terminal TT dinucleotide to create new recessed 3′ OH ends. The two 3′ OH groups then serve as nucleophiles in the joining reaction to attack the phosphate bonds of the cellular target DNA in a single-step transesterification to produce a gapped covalent intermediate with 2-nucleotide overhangs. Overhang removal, gap fill-in, and ligation to complete the integration are likely mediated by host repair mechanisms (2.Daniel R. Katz R.A. Skalka A.M. Science. 1999; 284: 644-647Crossref PubMed Scopus (224) Google Scholar), although participation by viral proteins has been suggested (3.Chow S.A. Vincent K.A. Ellison V. Brown P.O. Science. 1992; 255: 723-726Crossref PubMed Scopus (365) Google Scholar, 4.Sherman P.A. Dickson M.L. Fyfe J.A. J. Virol. 1992; 66: 3593-3601Crossref PubMed Google Scholar, 5.Kulkosky J. Katz R.A. Merkel G. Skalka A.M. Virology. 1995; 206: 448-456Crossref PubMed Scopus (73) Google Scholar). The final product is a 4-base pair shortened viral genome inserted in the host DNA flanked by 6-base pair inverted repeats derived from the six base pairs separating the sites of concerted joining. The CA near the ends of the viral long terminal repeats (LTR) is conserved among retroviruses, whereas the length of the flanking host repeats is virus-specific and is thought to be a structural consequence of a particular integrase (6.Hishinuma F. DeBona P.J. Astrin S. Skalka A.M. Cell. 1981; 23: 155-164Abstract Full Text PDF PubMed Scopus (55) Google Scholar, 7.Muesing M.A. Smith D.H. Cabradilla C.D. Benton C.V. Lasky L.A. Capon D.J. Nature. 1985; 313: 450-458Crossref PubMed Scopus (432) Google Scholar, 8.Starcich B. Ratner L. Josephs S.F. Okamoto T. Gallo R.C. Wong-Staal F. Science. 1985; 227: 538-540Crossref PubMed Scopus (67) Google Scholar, 9.Vink C. Groenink M. Elgersma Y. Fouchier R.A. Tersmette M. Plasterk R.H. J. Virol. 1990; 64: 5626-5627Crossref PubMed Google Scholar). For clarity, the remainder of this report will refer to the dinucleotide TT-trimming activity as the "processing" reaction and the subsequent sequence-dependent insertion of processed ends into a double-stranded DNA target as the "joining" reaction. The novel recombination activity specific to synapsed DNA substrates (10.Bao K. Skalka A.M. Wong I. J. Biol. Chem. 2002; 277: 12089-12098Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar) will be referred to as the "splicing" reaction. avian sarcoma virus integrase long terminal repeat absorbance at 260 nm Tris borate-EDTA The U5 and U3 regions in ASV LTRs include two separate nearly perfect inverted repeat sequences in these noncoding sections of the retroviral RNA genome. In the course of retroviral replication, the RNA genome is reverse-transcribed into a duplex DNA copy, and as a result of the strand-transfer mechanism of reverse transcriptase, the U5 and U3 sequences become the termini of the DNA genome (11.Peliska J.A. Benkovic S.J. Science. 1992; 258: 1112-1118Crossref PubMed Scopus (289) Google Scholar). Purified integrase along with Mn2+ or Mg2+ as a cofactor is sufficient to catalyze both processing and joining reactions in in vitro assays using synthetic oligodeoxynucleotide substrates with DNA sequences derived from the U3 and/or U5 viral LTR ends (12.Kukolj G. Skalka A.M. Genes Dev. 1995; 9: 2556-2567Crossref PubMed Scopus (38) Google Scholar, 13.Chow S.A. Methods. 1997; 12: 306-317Crossref PubMed Scopus (76) Google Scholar, 14.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). Typical in vitro integrase processing assays use radiolabeled substrates, with the reaction(s) allowed to proceed for 30–90 min before quenching since integrase shows low reactivity in such assays (13.Chow S.A. Methods. 1997; 12: 306-317Crossref PubMed Scopus (76) Google Scholar, 15.Scottoline B.P. Chow S. Ellison V. Brown P.O. Genes Dev. 1997; 11: 371-382Crossref PubMed Scopus (76) Google Scholar, 16.Pemberton I.K. Buckle M. Buc H. J. Biol. Chem. 1996; 271: 1498-1506Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). More recently, Vora and Grandgenett (17.Vora A. Grandgenett D.P. J. Virol. 2001; 75: 3556-3567Crossref PubMed Scopus (31) Google Scholar) studied the integrase-catalyzed joining reaction using an assay time of 5 min after a period of preincubation, aimed at "more closely reflecting the effects of the initial assembly events." In either case, the reaction products and unreacted substrates are separated by electrophoresis on agarose or denaturing polyacrylamide gels to resolve the dinucleotide-shortened processing products and the extended joining products from the original substrates. Subsequent quantitation of the two products is used to measure the extent of catalytic activity. Single-end substrate assays show poor processing efficiency, and they also fail to produce measurable amounts of concerted integration products (where, in the case of ASV, two viral ends are inserted six base pairs apart) as is observed with preintegration complexes purified from infected cells (18.Craigie R. Fujiwara T. Bushman F. Cell. 1990; 62: 829-837Abstract Full Text PDF PubMed Scopus (331) Google Scholar, 19.Katz R.A. Merkel G. Kulkosky J. Leis J. Skalka A.M. Cell. 1990; 63: 87-95Abstract Full Text PDF PubMed Scopus (297) Google Scholar). Consequently, products larger than the original substrate in these assays that use a substrate containing only one LTR-end-derived sequence are considered to be products of only half-reactions, as only one viral DNA end is joined to a target site (19.Katz R.A. Merkel G. Kulkosky J. Leis J. Skalka A.M. Cell. 1990; 63: 87-95Abstract Full Text PDF PubMed Scopus (297) Google Scholar). Assays involving substrates designed to resemble the linear retroviral genome, with an LTR end-derived sequence at either terminus, have resulted in >95% of the joining products coming from the recombination of two separate substrate molecules integrating into a single target molecule (14.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, 20.Aiyar A. Hindmarsh P. Skalka A.M. Leis J. J. Virol. 1996; 70: 3571-3580Crossref PubMed Google Scholar, 21.Fitzgerald M.L. Grandgenett D.P. J. Virol. 1994; 68: 4314-4321Crossref PubMed Google Scholar). Despite the lack of similarity to the nucleoprotein complex assembled in vivo, the assays involving such trimolecular reactions have been used to suggest the preference of integrase for a U3/U3 combination of ends over that of a U5/U5 combination, with the specificity for the biologically significant U3/U5 arrangement intermediately between the two (14.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). More recently, Brin and Leis (22.Brin E. Leis J. J. Biol. Chem. 2002; 277 (in press)Google Scholar), using a reconstituted HIV-1 integration system, demonstrated that concerted DNA integration requires the presence of both U3 and U5 ends in the donor DNA. DNase I footprint analysis of the assembled integrase-DNA complex required for integration has revealed that the region of protection at the U3 LTR end (∼20 base pairs) was at least twice that at the U5 LTR end (<10 base pairs), suggesting that the nucleoprotein complex is asymmetric when assembled in a fashion capable of full-site integration (17.Vora A. Grandgenett D.P. J. Virol. 2001; 75: 3556-3567Crossref PubMed Scopus (31) Google Scholar). In an attempt to mimic the geometric organization of the viral LTR ends of the in vivo preintegration complex at a more molecular level, Kukolj and Skalka (12.Kukolj G. Skalka A.M. Genes Dev. 1995; 9: 2556-2567Crossref PubMed Scopus (38) Google Scholar) designed a series of substrates that covalently linked two single-end substrates together in a head-to-head configuration using 1–3 nucleotides of single-stranded DNA (see Fig. 1A). It was hypothesized that these single-stranded tethers would provide sufficient flexibility to alleviate torsional or rotational strains arising from the structural alignment of the two viral ends bound within the integrase active site(s). By designing the substrates asymmetrically with respect to the length of the two ends and 5′ radiolabeling both ends, it was possible to quantitate processing products for both ends simultaneously in addition to what appeared to be extended joining products. Using in vitro integrase assays similar to those described above, these authors observed enhanced processing efficiencies with these synapsed-end substrates and concluded that the tether effectively brought together integrase subunits bound separately to the two cognate sites, thereby coordinating the formation of a requisite higher order oligomeric structure with enhanced activity. These observations were consistent with the suggestion by Murphy and Goff (23.Murphy J.E. Goff S.P. J. Virol. 1992; 66: 5092-5095Crossref PubMed Google Scholar) that integrase must recognize both DNA ends for efficient processing at either end to occur in vivo. Whereas the results with these synapsed substrates clearly illustrated the important relationship between assembly of an integrase multimer and the coordinated binding of both viral DNA ends, the assays were performed in the time regime where the enzyme-catalyzed reaction had undergone multiple turnovers. Although much effort has been expended to demonstrate that integrase functions as a true enzyme in its ability to catalyze multiple turnovers under steady-state conditions (24.Jones K.S. Coleman J. Merkel G.W. Laue T.M. Skalka A.M. J. Biol. Chem. 1992; 267: 16037-16040Abstract Full Text PDF PubMed Google Scholar), the physiological relevance of multiple turnover events is questionable considering that only a single round of catalysis is sufficient to achieve integration in vivo. To complete a first-turnover investigation of the processing reaction using synapsed-end substrates, 5′-5′ reverse-polarity substrates were designed (see Fig. 1B) that allow the simultaneous binding of two LTR ends at the active site. Additionally, these substrates were not susceptible to the integrase-catalyzed splicing reaction and consequently simplified the comparisons of the LTR ends. Analysis of presteady-state assays of these reverse-polarity substrates revealed that, although the U3 LTR sequence appears to be preferred by avian integrase when the termini are studied individually (25.Fitzgerald M.L. Vora A.C. Grandgenett D.P. Anal. Biochem. 1991; 196: 19-23Crossref PubMed Scopus (14) Google Scholar, 26.Vora A.C. Grandgenett D.P. J. Virol. 1995; 69: 7483-7488Crossref PubMed Google Scholar, 27.Fitzgerald M.L. Vora A.C. Zeh W.G. Grandgenett D.P. J. Virol. 1992; 66: 6257-6263Crossref PubMed Google Scholar, 28.Grandgenett D.P. Inman R.B. Vora A.C. Fitzgerald M.L. J. Virol. 1993; 67: 2628-2636Crossref PubMed Google Scholar), the U3/U5 combination of retroviral ends is the preferred substrate for integrase microscopically within a single turnover. Results from the first-turnover exponential analyses also have important bearing on previous interpretations of multiple-turnover product distributions. An examination of the exponential phases and their usefulness to a further understanding of the mechanism of integrase activity and the binding of substrates is also discussed. Oligodeoxyribonucleotides were synthesized by the Center for Gene Research and Biotechnology Central Services Laboratory (Oregon State University). Reversed-polarity oligodeoxyribonucleotides were synthesized using 5′-β-cyanoethyl phosphoramidites (Glen Research, Sterling, VA). Concentrations were determined spectrophotometrically in Tris-EDTA using the calculated extinction coefficients at 260 nm (29.Cantor C.R. Warshaw M.M. Shapiro H. Biopolymers. 1970; 9: 1059-1077Crossref PubMed Scopus (880) Google Scholar) listed in Table I.Table INomenclature and μm extinction coefficients at 260 nm DNA substratesNameaThe following is the naming convention used for annealed DNA substrates. Strands with sequences derived from the U5 and U3 ends of the ASV genome are designated with a "5" and "3," respectively. Strands containing the cognate sequence, CATT, are designated with a "t." Strands containing the complementary GTAA sequence are designated with a "b." Synapsed strands are designated with the length of the tether within parentheses. Duplex names consist of a concatenation of the names of all oligodeoxyribonucleotide strands annealed separated by slashes (/). Sequences labeled with 32P will be represented in the text with an asterisk (*) to denote 5′-end radiolabeling.Sequenceɛ2605t5′-GCTGAAGCAGAAGGCTTCATT-3′0.205b5′-AATGAAGCCTTCTGCTTCAGC-3′0.193t5′-GCTATTGCATAAGACTACATT-3′0.213b5′-AATGTAGTCTTATGCAATAGC-3′0.215b(2)3t5′-GCTATTGCATAAGACTACATTTAAATGAAGCCTTCTGCTTCAGC-3′0.435b(2)3b3′-CGATAACGTATTCTGATGTAAT-5′-5′-AAATGAAGCCTTCTGCTTCAGC-3′0.435b(2)5b3′-CGACTTCGTCTTCCGAAGTAAA-5′-5′-TAATGAAGCCTTCTGCTTCAGC-3′0.413b(2)3b3′-CGATAACGTATTCTGATGTAAT-5′-5′-AAATGTAGTCTTATGCAATAGC-3′0.44a The following is the naming convention used for annealed DNA substrates. Strands with sequences derived from the U5 and U3 ends of the ASV genome are designated with a "5" and "3," respectively. Strands containing the cognate sequence, CATT, are designated with a "t." Strands containing the complementary GTAA sequence are designated with a "b." Synapsed strands are designated with the length of the tether within parentheses. Duplex names consist of a concatenation of the names of all oligodeoxyribonucleotide strands annealed separated by slashes (/). Sequences labeled with 32P will be represented in the text with an asterisk (*) to denote 5′-end radiolabeling. Open table in a new tab The naming convention used for annealed DNA substrates is as follows. strands with sequences derived from the U5 and U3 ends of the ASV genome are designated with a "5" and "3," respectively; strands of duplex DNA containing ASV integrase cognate sequence, CATT, are designated with a "t"; strands containing the complementary GTAA sequence are designated with a "b"; synapsed strands are designated with the length of the tether within parentheses; duplexes are denoted as the concatenation of the names of the component single-stranded oligodeoxyribonucleotides separated by slashes (/); sequences 5′-end-radiolabeled with 32P will be specified in the text with an asterisk (*) at the beginning. The intensity of each product band, Ii(t), at each time, t, was first normalized with respect to the sum of intensities in the starting substrate band, I0(t), plus all product bands according to Equation 1 to determine the normalized product fraction Fi,norm(t). Fi,norm(t)=−Ii(t)I,(t)+∑i=1nIi(t)Equation 1 Fi,norm, was then corrected for background intensity present at t = 0 for the i th band and renormalized for background intensities of all product bands to obtain the final corrected product fraction, Fi,corr according to Equation 2. Fi,corr(t)=Fi,norm(t)−Fi,norm(0)1−∑i=1nFi,norm(0)Equation 2 Experimental time courses were fitted to Equation 3 consisting of n exponential terms, with amplitudes Ai and apparent rate constants λi plus a linear term with an apparent rate constant λlin to fit to the linear portion of the ensuing exponential phase. y=∑i=1nAi(1−e−λit)+λlintEquation 3 Non-linear least squares fittings were performed using Kaleidagraph software (Synergy, Redding, PA). These materials and protocols were identical to those described in detail in the first paper of this series (10.Bao K. Skalka A.M. Wong I. J. Biol. Chem. 2002; 277: 12089-12098Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). The normal-polarity substrate, used originally to develop the presteady-state assay for integrase (10.Bao K. Skalka A.M. Wong I. J. Biol. Chem. 2002; 277: 12089-12098Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar), positioned the cognate CATT of the U3 sequence internally (see Fig. 1A). As a result, this sequence became the site of a spurious site-specific splicing reaction (10.Bao K. Skalka A.M. Wong I. J. Biol. Chem. 2002; 277: 12089-12098Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). Because the site preference of the splicing reaction is identical to that of the processing reaction, i.e. the internal CATT, its presence interfered with accurate quantitation of enzymatic processing activity. A reverse-polarity substrate (Fig. 1B), incorporating a 5′-5′ reverse-polarity tether and containing the same sequence as that of the normal-polarity substrate (Table I), was therefore designed specifically to circumvent the splicing reaction while maintaining the advantages of tethering the viral LTR sequences (Fig. 1B). At high concentration of NaCl (400 mm), the activity of integrase with normal-polarity substrates was predominated by the splicing activity (Fig. 2A). To demonstrate that the reverse-polarity substrate is not susceptible to this splicing reaction, presteady-state assays comparing different DNA substrates with and without a tether were performed at high salt conditions favorable to the splicing reaction. Reactions were performed as described under "Experimental Procedures" with all substrates radiolabeled on *5t, a 21-mer that is endonucleolytically cleaved by integrase at the normal processing, minus 2 position and at a second, minus 3 position to yield radiolabeled 19- and 18-mer products. The splicing reaction would yield a 46-mer product with the normal-polarity substrate *5t/5b(2)3t/3b and an anticipated 23/24-mer with the reverse-polarity substrate *5t/5b(2)3b/3t. Fig. 2A shows that the major product, at 400 mmNaCl, with the normal-polarity substrate was the 46-mer splicing product, whereas the 19-mer processing and shorter products were present in minor but detectable amounts. In contrast, reactions performed under identical conditions with the reverse-polarity substrate, *5t/3b(2)5b/3t, yielded predominantly processing products (19- and 18-mers) (Fig. 2B). Integrase-mediated splicing activity of this reverse-polarity substrate would have resulted in +2 and +3 products of 23- and 24-nucleotide lengths, respectively; products of such sizes accumulated in minute amounts and only at the two longest reaction times examined (1200 and 1800 s). By comparison, the processing products appeared within 5–10 s and in much greater quantities. These data demonstrated that even under reaction conditions chosen specifically to favor splicing over processing, the 5′-5′ tethering preferentially mediates integrase-catalyzed processing. To rule out the possibility that the increased NaCl concentration itself was capable of having promoted the initial exponential phase of processing activity with the reverse-polarity substrate, a control experiment was performed with an unsynapsed, single-ended substrate *5t/5b. Fig. 2C shows clearly the lack of any detectable integrase activity with this substrate under these conditions. Even at the longest time point assayed, 1800 s, *5t/5b was minimally processed. These results showed that without the scaffolding provided by the tethering of the two ends, integrase was unable to utilize the cognate sequence for enzymatic activity at this higher NaCl concentration. Fig. 3A shows the results from a typical first-turnover experiment performed as described under "Experimental Procedures." Assay results with synapsed 5t/5b(2)3b/*3t and unsynapsed *3t/3b substrates (see Table I) are shown to illustrate the effect of tethering two viral end sequences in a head-to-head manner via a 5′-5′ linkage. The radiolabeled 21-mer, *3t, with a sequence corresponding to the viral U3 LTR, used in both substrates, contained the 3′ terminal cognate sequence CATT-OH. In the processing reaction, this sequence was endonucleolytically cleaved by integrase at the minus 2 position to yield a 19-mer and a TT dinucleotide as the major products. Additionally, an 18-mer minor product was also observed that is consistent with the less specific processing activity at the minus 3 position observed when Mn2+ is used as the metal cofactor (19.Katz R.A. Merkel G. Kulkosky J. Leis J. Skalka A.M. Cell. 1990; 63: 87-95Abstract Full Text PDF PubMed Scopus (297) Google Scholar, 30.Katzman M. Katz R.A. Skalka A.M. Leis J. J. Virol. 1989; 63: 5319-5327Crossref PubMed Google Scholar, 31.Terry R. Soltis D.A. Katzman M. Cobrinik D. Leis J. Skalka A.M. J. Virol. 1988; 62: 2358-2365Crossref PubMed Google Scholar). Both 19- and 18-mer products were summed and plotted to quantitate total integrase-catalyzed processing activity. Kinetic experiments revealed multi-phasic reactions at all integrase concentrations tested. The time-dependent appearance of the processing product was best described by a series of presteady-state exponential phases followed by a linear phase according to Equation 3. The number of exponential phases observed was dependent on both the substrate used and the duration of time course examined. For all time courses, we have chosen to report the model-independent, actual molar concentration of product observed without further interpretation for accuracy. Compared with an unsynapsed substrate (*3t/3b), the initial exponential product formation phase of the synapsed substrate (5t/5b(2)3b/*3t) reaction occurred with larger amplitude and a significantly faster rate. The difference in burst amplitudes and rate constants for the synapsed versus unsynapsed substrates directly reflected differences in the extent of productive complex formation between enzyme and DNA during the preincubation period (before the initiation of the reaction with Mn2+). Furthermore, the synapsed substrate contained two CATT-OH cognate sites at which processing could have occurred, one at the 3′-end of the 3t strand and one at the 3′-end of the 5t strand. Because only the 3t strand was radiolabeled, however, processing at the cognate site of the U5 sequence was silent in these assays. As a consequence, the amplitude measured for the synapsed substrate did not include the amount of productive complexes formed at the U5 cognate site, and the large difference in amplitudes observed in this experiment actually underestimated the true enhancement of productive nucleoprotein complex formation attributable to the tethering of the two viral LTR ends. At time points extended beyond the early exponential phases, both time courses paralleled each other for the synapsed and unsynapsed substrates, indicating similar rates of reaction in subsequent steps of the enzyme. Experiments performed using radiolabeled *5t showed similar but lower reactivity under the same conditions. To assess the effect of enzyme concentration on the differences in reactivities of the two substrates, titrations of enzyme concentration were performed for both substrates. Typical results are shown in Figs.3, B and C, for assays conducted at integrase concentrations varying from 0.5 to 5.0 μm and a substrate concentration of 0.5 μm. The data were fit to Equation 3, and the best fit amplitudes and rate constants of the initial phase were plotted as a function of integrase concentrations. In the case of *3t/3b (closed squares, solid lines), both the apparent rate constant, λ1, and amplitude, A1, increased linearly with increasing integrase concentration with slopes of 0.0287 (r = 0.99) and 0.0049 (r = 0.99), respectively. In contrast, although the amplitude of the initial exponential phase in the synapsed substrate reaction increased with increasing protein concentration (with the exception of the highest protein concentration where protein aggregation becomes a problem), the rate constant for the initial exponential phase remained 0.2 s−1, independent of integrase concentration for the range of concentrations tested. In contrast, the single-end substrate only approached this rate at the highest integrase concentration range. These data show that the lower limit of the rate constant for the processing reaction is 0.2 s−1. The reverse-polarity substrates allowed exclusive examination of the processing activity and, therefore, made possible the direct comparison of the reactivities of the U5 and U3 sequences. The reactivities of all four possible combinations of synapsed end sequences, U3/U3, U3/U5, U5/U5, and U5/U3, were separately measured using *3t/3b(2)3b/*3t, 5t/5b(2)3b/*3t, *5t/3b(2)5b/*5t, and *5t/5b(2)3b/3t, respectively. The results from presteady-state assays, as described under in "Experimental Procedures," were obtained and compared over a range of both NaCl (130–500 mm) and integrase (0.5–20 μm) concentrations. Fig. 4A shows a typical direct comparison of the time-dependent appearance of processing products of the U3 end in the context of a synapsed U3/U3 pair (open circles, *3t/3b(2)3b/*3t) versus that of the same U3 sequence in a synapsed U3/U5 combination (closed circles, 5t/5b(2)3b/*3t). At 5 μmintegrase, 0.5 μm reverse-polarity substrate DNA, and 130 mm NaCl, the best fits of the time-dependent appearance of processing products were characterized by two initial exponential phases followed by a slower linear phase representing the beginning of a third exponential phase. However, because the substrate used to assay the U3/U3 combination, *3t/3b(2)5b/*3t, contained two radiolabeled 21-mers, whereas 5t/5b(2)3b/*3t was radiolabeled on only one DNA strand, it was necessary to divide the best fit of the U3/U3 data by two. Comparison of the adjusted curve (dotted curve) with the best fit of the data from the U3/U5 substrate (solid curve) revealed that the U3 end is processed to a greater extent when bound concurrently with a U5 end than when bound with another U3 sequence. Similarly, Fig. 4B shows the results from parallel presteady-state experiments comparing the reactivity of the U5 end in the context of either a U5/U5 pair (open squares, *5t/3b(2)5b/*5t) or a U5/U3 combination of ends (closed squares, *5t/3b(2)5b/3t). As described above, the