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J. Biol. Chem., Vol. 281, Issue 32, 22707-22719, August 11, 2006

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Relationship between the Oligomeric Status of HIV-1 Integrase on DNA and Enzymatic Activity^*

Elvire Guiot, Kevin Carayon, Olivier Delelis, Françoise Simon, Patrick Tauc, Evgenii Zubin, Marina Gottikh, Jean-François Mouscadet, Jean-Claude Brochon, and Eric Deprez¹

From the Laboratoire de Biotechnologie et Pharmacologie Genetique Appliquee, CNRS, UMR8113, Ecole Normale Supérieure de Cachan, 61 av du Président Wilson, 94235 Cachan, France and the Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119992 Moscow, Russia

Received for publication, March 8, 2006 , and in revised form, May 24, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The 3'-processing of the extremities of viral DNA is the first of two reactions catalyzed by HIV-1 integrase (IN). High order IN multimers (tetramers) are required for complete integration, but it remains unclear which oligomer is responsible for the 3'-processing reaction. Moreover, IN tends to aggregate, and it is unknown whether the polymerization or aggregation of this enzyme on DNA is detrimental or beneficial for activity. We have developed a fluorescence assay based on anisotropy for monitoring release of the terminal dinucleotide product in real-time. Because the initial anisotropy value obtained after DNA binding and before catalysis depends on the fractional saturation of DNA sites and the size of IN·DNA complexes, this approach can be used to study the relationship between activity and binding/multimerization parameters in the same assay. By increasing the IN:DNA ratio, we found that the anisotropy increased but the 3'-processing activity displayed a characteristic bell-shaped behavior. The anisotropy values obtained in the first phase were predictive of subsequent activity and accounted for the number of complexes. Interestingly, activity peaked and then decreased in the second phase, whereas anisotropy continued to increase. Time-resolved fluorescence anisotropy studies showed that the most competent form for catalysis corresponds to a dimer bound to one viral DNA end, whereas higher order complexes such as aggregates predominate during the second phase when activity drops off. We conclude that a single IN dimer at each extremity of viral DNA molecules is required for 3'-processing, with a dimer of dimers responsible for the subsequent full integration.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The integration of a DNA copy of the HIV-1² genome into the host genome is a crucial step in the life cycle of the retrovirus. Integrase (IN) is responsible for the two consecutive reactions that constitute the overall integration process. The first of these two reactions is 3'-processing, which involves cleavage of the 3'-terminal GT dinucleotide at each extremity of the viral DNA. The hydroxyl groups of newly recessed 3'-ends are then used in the second reaction, strand transfer, for the covalent joining of viral and target DNAs, resulting in full-site integration. IN is sufficient for catalysis of the 3'-processing reaction in vitro, using short-length oligodeoxynucleotides (ODNs) that mimic one viral long terminal repeat (LTR) in the presence of the metallic cofactor Mg²⁺. This reaction generates two products: the viral DNA containing the recessed extremity and the GT dinucleotide. One of the two products, the processed viral DNA, as well as the target DNA serve as substrates for the subsequent joining reaction.

IN belongs to the superfamily of polynucleotidyl transferases. Its catalytic core domain contains a triad of acidic residues constituting the D,D-35-E motif, which is strictly required for catalysis. The catalytic core establishes specific contacts with the viral DNA and, together with the C-terminal domain, is involved in DNA binding (1–4). The 3'-processing reaction is highly specific, and the terminal 13 bp of the LTR play a key role for Mg²⁺-dependent 3'-processing in terms of reaction specificity (4, 5). In particular, the CA sequence preceding the GT dinucleotide cleaved by IN is strictly required. Reaction specificity seems to depend on the catalytic step, because no significant difference in affinity is observed in vitro for different DNA sequences (2, 3, 5, 6). The catalytic mechanism of IN has been extensively studied, but the structural determinants of IN activity remain unclear, and controversy remains concerning the multimeric status of active IN. Single- and double-domain crystallographic structures have been produced for IN, alone or complexed with the IN-binding domain of the cellular partner lens epithelium-derived growth factor, and all these structures display a conserved dimeric structure of the catalytic core (7–11). Based on topological considerations concerning the distance separating the active sites of the two protomers, which is more than double the distance separating the two joining sites on the target DNA (separated by 5 bp), a multimeric state of an order higher than dimers, tetramers at least, is required for complete integration. However, it remains unclear whether such a higher order multimeric state is also required for 3'-processing activity and how this activity is modulated by the self-association properties of IN. Moreover, most enzymatic studies are carried out with an excess of IN over DNA substrate. Taking into account the low solubility of IN, we investigated whether high order multimers or aggregated forms of IN favored by high protein concentrations in solution were detrimental or beneficial for activity.

We developed an assay for monitoring DNA binding and subsequent 3'-processing in the same sample. This assay makes possible the separation of binding and catalytic parameters and the study of real-time kinetics. It is based on steady-state fluorescence anisotropy (r), which is sensitive to rotational diffusion, and thus suitable for studies aiming to identify structural modifications leading to a significant change in molecular size (12–15). Using a fluorescent probe covalently linked to the GT dinucleotide, this makes it possible to follow DNA binding and dinucleotide release, because both steps strongly influence molecular size of the fluorescent moiety. The anisotropy-based technique is also highly suitable for studies of the relationship between the overall size of IN·DNA complexes and activity. We found that, for low IN:DNA ratios, the r values obtained after DNA binding and before catalysis were fully predictive of subsequent IN activity, according to the fractional saturation function. For high IN:DNA ratios, anisotropy continued to increase, but 3'-processing activity decreased. Because r depends on both fractional saturation and the molecular size of complexes at saturation of DNA sites, our results show that high order multimers or aggregated states of IN are detrimental to 3'-processing activity. Activity levels were highest for non-aggregative smaller species. A more precise characterization of catalytically competent complexes by time-resolved fluorescence anisotropy (TFA) (16–20), confirmed steady-state data and led to the identification of dimeric forms as the most active forms for 3'-processing. Our results also highlight that only the IN:DNA ratio but not the IN concentration per se determines the aggregation properties and thus the activity of IN and that DNA binding stimulates the self-organization of IN to give a catalytically competent non-aggregative form.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Oligonucleotides and Nomenclature—Unlabeled and fluorescein-labeled single-stranded (ss) ODNs were purchased from Eurogentec (Liege, Belgium) (except for the 2'-aminouridine-containing ODN) and purified by electrophoresis in acrylamide gels. The specific HIV sequence was 5'-GTGTGGAAAATCTCTAGCAGT-3' (the bases removed by IN are underlined). This sequence was denoted a, and the complementary nonprocessed strand was denoted b. Fluorescein (F) was attached at the 5'- or the 3'-end of strand a or b, via a 6-carbon linker. As an example, the specific double-stranded (ds) ODN used to monitor activity was called HIV-a3F, indicating that fluorescein was attached to the 3'-end of strand a. HIV-GTGT-a3F was identical except that the four terminal bases at the 3'-end of strand a were GTGT rather than the canonical sequence CAGT. The nonspecific sequence, NS-TTCC, was 5'-ACCTATGCGCCGCTAGATTCC-3' (strand a). Two other nonspecific ODNs with different 3'-ends of strand a were derived: NS-CACC and NS-CAGT. ds ODNs were obtained by mixing equimolar amounts of complementary strands in 20 mM Tris-HCl (pH 7.2), 100 mM NaCl. The mixture was heated to 85 °C for 5 min, and annealing was allowed by slow cooling to 25 °C.

The uridine-containing ODN (corresponding to strand a of the DNA substrate HIV-aUF) contains a fluorescein attached to the 2'-amino group of the 3'-terminal 2'-aminouridine. It was synthesized as follows: the solid support was prepared as previously described (21). Briefly, 2'-deoxy-5'-O-4,4'-dimethoxytrityl-2'-O-trifluoracetamidouridine (0.2 mmol) was co-evaporated with pyridine (3 x 5 ml) and dissolved in dry pyridine (2 ml). Succinylated long-chain alkylamine-controlled pore glass (500 Å) (200 mg), 2,4,6-triisopropylbenzenesulfonyl chloride (0.6 mmol), and 1-methylimidazole (1.2 mmol) were added, and the mixture was incubated for 3 h at 25°C. The support was then filtered and washed successively with pyridine, CH₂Cl₂, and ether. Starting from 2'-deoxy-5'-O-4,4'-dimethoxytrityl-2'-O-trifluoracetamidouridine-derivatized long-chain alkylamine-controlled pore glass (500 Å), a 21-mer ODN was assembled on an ABI394B DNA synthesizer, by the phosphoramidite method, according to the manufacturer's recommendation. Protected 2'-O-deoxyribonucleoside phosphoramidites and S-ethylthiotetrazole were purchased from Glen Research. Ammonia was used for cleavage from the support and deprotection overnight at 55 °C. The reaction mixture was then analyzed by reverse-phase high-performance liquid chromatography in ion-pair mode. For fluorescein labeling, 1 mg of fluorescein isothiocyanate dissolved in 20 µl of dimethylformamide was added to a solution of 40 nmol of 2'-amino ODN in 130 µl of sodium carbonate-bicarbonate (1 M, pH 9):water (v/v, 5:8) (22, 23). The mixture was incubated at 25 °C in the dark for 18 h and then loaded onto an NAP5-Sephadex G-25 column (Amersham Biosciences) pre-equilibrated in water. The ODN was eluted with water and analyzed by reversed-phase high-performance liquid chromatography in ion-pair mode.

IN Purification and Standard Analysis of the 3'-Processing Reaction—HIV-1 IN (32 kDa) was purified under native conditions, as previously described (24). Standard IN assay, using ³²P-labeled ODN and gel electrophoresis, was carried out as previously described (24). Gels were analyzed on a STORM 840^TM PhosphorImager (Amersham Biosciences) and quantified with ImageQuaNT^TM 4.1 software. The 3'-processing activity was calculated as follows: activity (%) = 19-mer/(21-mer + 19-mer) x 100.

Steady-state Fluorescence Anisotropy Assay—Steady-state anisotropy values were recorded on a Beacon 2000 instrument (PanVera, Madison, WI), in a cell thermostatically held at 25 or 37 °C, for the DNA-binding step and 3'-processing reaction, respectively. Unless otherwise stated, we studied the formation of IN·DNA complexes by incubating fluorescein-labeled ds ODNs with IN in 20 mM Tris (pH 7.2), 1 mM dithiothreitol, 20 mM NaCl, 5 mM MgCl₂ (the sample volume was 200 µl). The fractional saturation function (Y) was calculated as follows,

(Eq. 1)

where r_max and r_ODN are the anisotropies of IN-bound and free ODN, respectively (no significant concomitant change in fluorescence intensity was observed). After the DNA-binding step, the temperature was raised from 25 to 37 °C for monitoring of the catalytic process. The 3'-processing activity was assessed by quantifying the decrease in r. Two independent methods were used for quantification as follows. (i) In fixed-time experiments, the reaction was stopped by adding SDS (0.25% final), disrupting all the IN·DNA complexes in the sample. In such experiments, the solution contained two fluorescent species: the non-processed ODN and the fluorescein-labeled dinucleotide released by the cleavage reaction. The fraction of dinucleotides released (F_dinu = [GT]/[DNA]_total) is given by Equation 2,

(Eq. 2)

where r_NP and r_dinu are the anisotropy values for pure solutions of non-processed ds ODN and dinucleotide, respectively (fluorescence did not change significantly during the reaction). We used the 5'-GT-3'F dinucleotide (Eurogentec) to determine r_dinu. (ii) In real-time conditions, an additional fluorescent population corresponding to IN complexed with the unprocessed ds ODN, is present in the sample. In this case, F_dinu was calculated as follows,

(Eq. 3)

where r_max is the characteristic r value obtained for optimal activity, and r_{t = 0} is the r value obtained at the end of the DNA-binding step (before the start of the reaction). The 3'-processing activity obtained with Equations 2 and 3 is referred to as Activity_SDS and Activity_real-time, respectively. Activity_real-time was not used if r_{t = 0} was higher than 0.22 (aggregation of IN on DNA not negligible). We analyzed single-turnover kinetics using the Equations 4 and 5,

(Eq. 4)

with

(Eq. 5)

where k_chemistry is the single-turnover rate constant, and K_d,app is the apparent K_d (25). Reactions were conducted in the presence of Mg²⁺ (not Mn²⁺) to limit nonspecific hydrolysis products (cleavages at positions –3, –4, etc.), because anisotropy cannot discriminate between these small products (which are minor products with Mg²⁺ but not with Mn²⁺) and the specific GT product. The r values for the TAMRA-labeled DNA substrate (_ex = 562 nm and _em = 582 nm) were recorded on a Cary Eclipse spectrofluorometer (Varian, Mulgrave, Australia) in polarization mode and were compared with values obtained with the same instrument and HIV-a3F as the substrate.

K_d,app as a function of ODN length were determined by competition experiments. Fluorescein-labeled ds ODN HIV-a5F (4 nM) was preincubated with various concentrations of unlabeled ss or ds HIV ODN (from 10- to 45-mers) in 20 mM Tris buffer (pH 7.2) supplemented with 1 mM dithiothreitol, 20 mM NaCl, and 5 mM MgCl₂. IN (150 nM final concentration) was then added, and r values were recorded. r (= r–r_ODN) was plotted against competitor concentration to determine K_d,app (concentration of competitor decreasing the initial r value by 50%).

Time-resolved Fluorescence Experiments—Time-resolved fluorescence parameters (lifetimes and correlation times) were obtained from the two polarized fluorescence decays I_||(t) and I_||(t), using the time-correlated single photon counting technique. The instrumentation setup was essentially similar to those previously described (16, 17), with modifications: the time scaling was 19.5 ps per channel and 4096 channels were used. The excitation light pulse source was a Ti:sapphire laser (Millennia-pumped Tsunami femtosecond laser, Spectra Physics) (repetition rate: 8 MHz) associated with a second harmonic generator tuned to 490 nm. The emission monochromator (ARC SpectraPro-150) was set to 530 nm ( = 15 nm). The two polarized components were collected alternately over a period of 30 s (total count of I_||: 15,000,000). The reaction mixture contained 20 mM Tris, pH 7.2, 20 mM NaCl, 5 mM MgCl₂, 1 mM dithiothreitol. The correlation time () distributions of free ss or ds ODNs were obtained at ODN concentrations of 10 nM or 0.5 µM. All ODNs (from 10- to 45-mer) mimicked the U5-end of the HIV-1 DNA and were fluorescein-labeled at the 5'-end of strand a. IN·DNA complexes were analyzed using ds HIV-a5F and various IN:DNA ratios, from 40 to 400. We analyzed both decays, I_||(t) and I_||(t), by the maximum entropy method (26). Fluorescence anisotropy decay is described by Equations 6 and 7,

(Eq. 6)

with

(Eq. 7)

where _i is the individual rotational correlation time, and _i is the associated amplitude. ₀ was found to lie between 0.36 and 0.37. Normalization of for a given temperature was performed using,

(Eq. 8)

where is the viscosity, V is the volume of the rotating unit, k is the Boltzmann constant, and T is the temperature (K).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Monitoring 3'-Processing Activity by Steady-state Fluorescence Anisotropy—Fluorescence anisotropy measurements are based on the principle of photoselective excitation of a fluorophore by a polarized light, providing information about rotational motions of the fluorophore or fluorescently labeled molecule between photon absorption and emission. Some events such as overall rotational diffusion or flexibility are major causes of light depolarization. High levels of anisotropy are generally associated with large molecules or complexes characterized by slow rotational diffusion or low flexibility level. In this study, we used an extrinsic fluorophore covalently linked to DNA to monitor the binding of IN to viral DNA substrate and the subsequent 3'-processing reaction, in the same assay. Both DNA binding and 3'-processing would be expected to have a significant effect on the anisotropy parameter, because each of these steps has a major effect on the molecular size of the fluorescent moiety. The principle underlying the anisotropy-based assay is summarized in Fig. 1. The binding of IN to a ds ODN mimicking one end of the viral DNA increases the steady-state anisotropy value (r), most likely by restricting flexibility, allowing the calculation of fractional saturation function. Following DNA binding, the same sample can be shifted to a permissive temperature for 3'-processing activity. When the fluorophore is linked to the 3'-terminal GT dinucleotide, release of the dinucleotide product should significantly decrease r. The fraction of dinucleotides released can be determined from the real-time decrease in r observed during the reaction (r_real-time, corresponding to the difference between IN·DNA complex and free dinucleotide) or after the disruption of all IN·DNA complexes by the addition of SDS (r_SDS, corresponding to the difference between unprocessed ds ODN and free dinucleotide). Unlike most standard 3'-processing assays, which are based on quantification of the processed strand (first reaction product), the anisotropy-based assay monitors the released dinucleotide (second reaction product).

View larger version (17K): [in this window] [in a new window]   FIGURE 1. Principle of the activity assay based on fluorescence anisotropy. The substrate is a 21-bp HIV ODN labeled with fluorescein at the 3'-end of the CAGT sequence. Upon IN binding to ODN, steady-state anisotropy (r) increases. At 37 °C, release of the terminal fluorescein-labeled GT dinucleotide by the 3'-processing reaction leads to a decrease in r. Activity can then be quantified by two methods: 1) The fraction of released dinucleotides (Fdinu) is related to the amplitude r and can be estimated from the direct observation of decreases in r values during the reaction (real-time assay). 2) The reaction can be quenched at any time by adding SDS, which disrupts the IN-ODN complexes (fixed-time assay). In this case, Fdinu is related to the amplitude rSDS.

View larger version (16K): [in this window] [in a new window]   FIGURE 2. The decrease in steady-state fluorescence anisotropy during 3'-processing is dependent on the position of fluorescein on the DNA substrate. A, position of the fluorophore on the various ODNs. Fluorescein was attached to the 3'- or 5'-extremity of each strand (a or b, where a is the strand containing the specific CAGT terminal sequence) giving HIV-a3F, HIV-a5F, HIV-b3F, or HIV-b5F. HIV-aUF has a fluorescein-labeled uridine at the 3'-terminal position of strand a. The 3'-processing reaction involves the cleavage of the terminal GT dinucleotide, as indicated by the arrow. IN was allowed to bind to the various ODNs for 20 min at 25 °C (3'-processing was undetectable during this preincubation step). The samples were then incubated at 37 °C, and r values were recorded over 4 h. r (B) or r (%) (C) was plotted against time. r (%) is a relative r value calculated as follows: r (%) = (r – rODN)/(rt = 0 – rODN) x 100. White squares: HIV-a3F, no Mg2+; black circles: HIV-a3F, 5 mM Mg2+; white circles: HIV-b5F, 5 mM Mg2+; black squares: HIV-a5F, 5 mM Mg2+; black triangles: HIV-b3F, 5 mM Mg2+; and white triangles: HIV-aUF, 5 mM Mg2+. IN and ODN concentrations were 120 and 4 nM, respectively.

The 3'-processing reaction is strongly sequence-dependent, and the endonucleolytic site includes the crucial conserved CA dinucleotide immediately preceding the GT dinucleotide. We assessed sequence specificity, using the anisotropy-based assay and the following ODNs: HIV-a3F corresponds to the wild-type (wt) sequence, whereas HIV-GTGT-a3F is a variant in which the 3'-terminal CAGT sequence is replaced by GTGT. We also tested three nonspecific sequences, NS-TTCC-a3F, NS-CACC-a3F, and NS-CAGT-a3F. We found that the DNA-binding step was not influenced by sequence. Indeed, the final r values obtained after DNA binding were similar for all ODNs, irrespective of sequence context, confirming that in vitro assays primarily reveal the nonspecific DNA binding mode of IN. In contrast, sequence had a major effect on r value in catalysis conditions (Fig. 3A). Only HIV-a3F gave a large decrease in r. This decrease was abolished by replacement of the 3'-terminal sequence CAGT by GTGT or the use of nonspecific sequences, even with the CAGT sequence at the 3'-end. These results confirm that the CA dinucleotide is strictly required but not sufficient for activity, particularly in the presence of Mg²⁺, which gives more stringent conditions than Mn²⁺ (4, 5). We also investigated the effect of a styrylquinoline compound, FZ41, which inhibits 3'-processing activity (30). The anisotropy-based approach has been shown to be suitable for assays of the effects of inhibitors on the formation and stability of IN·DNA complexes, independently of catalysis, and has been successfully used in studies of competitive inhibitors preventing IN·DNA interactions (12). Here, we investigated both steps, DNA binding and catalysis, in the presence of inhibitor. The IC₅₀ value for 3'-processing was found to be 0.74 µM (Fig. 3B), consistent with the value determined by standard gelelectrophoresis (30). In the same experiment, the study of the DNA-binding step revealed the competitive nature of FZ41: r_{t = 0} decreased with increasing FZ41 concentration (not shown), suggesting that FZ41 primarily prevents the IN-DNA interaction.

View larger version (13K): [in this window] [in a new window]   FIGURE 3. Assessment of the specificity of the 3'-processing reaction as monitored by the anisotropy-based method. A, sequence-specific decrease in steady-state anisotropy. The reaction was performed in the presence of Mg2+ with various DNA substrates. r (%) was plotted against time (see the legend for Fig. 2). All the ODNs tested were fluorescein-labeled on the 3'-extremity of strand a. HIV-a3F (black circles) corresponds to the wt sequence, whereas HIV-GTGT-a3F (white triangles) is a variant in which the CAGT 3'-terminal sequence was replaced by GTGT. Three nonspecific sequences (NS) were also studied (see "Experimental Procedures") and differed in the last four bases at the 3'-end, which are explicitly mentioned in their names: NS-TTCC-a3F (black squares), NS-CACC-a3F (white circles), NS-CAGT-a3F (white squares). The experimental conditions were similar to those described in the legend for Fig. 2. B, inhibition of 3'-processing activity by FZ41 compound, as monitored by fluorescence anisotropy. Activity calculations were based on measurements of rSDS.

View larger version (29K): [in this window] [in a new window]   FIGURE 4. 3'-Processing activity of IN. A, time course of 3'-processing activity, as obtained with the real-time assay. The DNA substrate was HIV-a3F (4 nM) in the presence of 180 nM IN and 5 mM Mg2+. Activity was calculated directly from the decrease in r values recorded at 37 °C (as shown in Fig. 2B). This activity was named "Activityreal-time" in contrast to "ActivitySDS," which was calculated from rSDS when all the native IN·DNA complexes were disrupted by SDS after various incubation times. B, correlation between Activityreal-time and ActivitySDS values. The various activity values were obtained by varying either the incubation time or the IN:DNA ratio. We used r as measured under real-time conditions or after adding SDS to calculate the fraction of released dinucleotides, giving Activityreal-time or ActivitySDS, respectively. The linear fit gives a slope of 0.97 (R2 = 0.96). C, 3'-processing activity, as detected by radioactivity on a denaturing acrylamide/urea gel. Left, non-fluorescent DNA substrate. Right, fluorescein-labeled DNA substrate (HIV-a3F). The upper and lower bands correspond to the 21-mer DNA substrate and the 19-mer processed DNA product, respectively. The slower migration of HIV-a3F is due to the presence of fluorescein at the 3'-extremity.

The Initial Steady-state Anisotropy Value Is Not Predictive of 3'-Processing Activity—The final r value obtained after the DNA-binding step corresponds to the initial r value for activity (r_{t = 0}) and is related, in first approximation, to the fractional saturation function (Y). Under single-turnover conditions, activity is expected to increase with Y, and the fraction of released dinucleotides should therefore be proportional to the number of IN·DNA complexes initially formed in the sample. Interestingly, a plot of 3'-processing activity against r_{t = 0} gave a clear bell-shaped curve (Fig. 5A). The various r_{t = 0} values were obtained by varying the initial IN:DNA ratio from 4.2 to 400, corresponding to different mixtures of IN (from 50 to 800 nM) and ODN (from 2 to 12 nM). The optimal ratio was found to be 40 (Fig. 5B) and corresponds to an r_{t = 0} value of 0.226. Larger values (up to 0.272) led to a significantly decrease in the 3'-processing activity. Increases in r_{t = 0} may occur for two reasons: (i) the number of free DNA molecules decreases with increasing IN:DNA ratio and (ii) self-assembly of IN on DNA may further increase the size of the complexes. The first phase (r_{t = 0} < 0.226) most likely corresponds to an increase in the number of complexes, increasing both r_{t = 0} and product formation. In the second phase (r_{t = 0} > 0.226), the activity drops off suggesting that high order multimeric forms of IN on DNA, possibly aggregates, formed under conditions of high IN:DNA ratio and characterized by high r values, are less active than lower order multimeric forms. Furthermore, the r_max value corresponding to saturation ([DNA]_free 0) must be below 0.272, and, thus, 0.226 is the best estimate for r_max.

View larger version (21K): [in this window] [in a new window]   FIGURE 5. 3'-Processing activity as a function of initial steady-state anisotropy. Various mixtures of IN (from 50 to 800 nM) and HIV-a3F DNA substrate (from 2 to 12 nM), giving different IN:DNA ratios, were incubated in the presence of Mg2+ for 20 min at 25 °C until r reached a plateau. Samples were then shifted to 37 °C and rt = 0 (=anisotropy value before the start of the reaction) was recorded. We calculated 3'-processing activity from rSDS values (at t = 400 min) and plotted this activity against rt = 0 (A) or IN:DNA ratio (B).

Characterization of the IN_act·DNA and IN_agg·DNA Complexes Using TFA—Under site saturation conditions, r values reflect the molecular size of the complexes in a purely indicative manner. We evaluated the molecular sizes of active IN·DNA complexes in greater detail by studying representative samples corresponding to different r values, as shown in Fig. 5A, independently by TFA. This approach allows the determination of rotational correlation times (), which are directly related to hydrodynamic volume according to Equation 8. We first characterized ss and ds ODNs of various lengths (10- to 45-mer) in the absence of IN (Table 1). All ODNs were characterized by short values (₁ and ₂), which can reasonably be assigned to rotation of the fluorescein moiety around the linker at the 5'-end of the DNA and the flexibility of the linker itself. Unlike ₁ and ₂, longer values (₃ and ₄) strongly depend on length and thus account for the overall rotational motion of the ODN. For each length, the longest correlation time was 2–2.7 times longer for ds than for ss ODNs. For example, the ₄ values for the 21-mer ODN were 4.3 and 8.6 ns for ss and ds molecules, respectively. We found that the 21-mer ds ODNs HIV-a5F, HIV-b3F, HIV-b5F, NS-TTCC-a3F, and NS-CACC-a3F displayed similar distributions and were characterized by r values of 0.045–0.055 (data not shown). In contrast, the 21-mer ds ODNs HIV-a3F, HIV-aUF, HIV-GTGT-a3F, and NS-CAGT-a3F, which had higher r values (0.105), gave similar correlation times to the previous series, but with different _i/₀ ratios (data not shown). For example, HIV-a3F had a long ₄ (9.6 ns), similar to that of HIV-a5F (8.6 ns), but the flexibility of the fluorophore was significantly lower, as demonstrated by the higher ₄/₀ ratio (= 0.5) than for HIV-a5F (₄/₀ = 0.125) (the fractional amplitude of fast motions is lower for HIV-a3F than for HIV-a5F). This decrease in flexibility seems to be related to the presence of a 3'-terminal GT and accounts for the higher r value (similar results were obtained using TAMRA; data not shown). Finally, the 21-mer ODN was the most suitable substrate for studies of IN·DNA complexes by TFA, because longer ODNs (36 or 45 bp) had a long , which could complicate the analysis of IN·DNA complexes. Moreover, shorter ODNs (10 or 13 bp) were not chosen for reasons of affinity, because we found that ODN length had a strong effect on K_d,app (Fig. 7).

View this table: [in this window] [in a new window]   TABLE 1 Correlation times () obtained for fluorescein-labeled ODNs of various lengths at 25 °C

View larger version (25K): [in this window] [in a new window]   FIGURE 6. Product formation is related to the initial fractional saturation function. A, 3'-processing kinetics for different IN:DNA ratios. Activity was measured using different ratios (below 40:1, rt = 0 < 0.22, to limit aggregation). HIV-a3F concentration was 4 nM with 10 (black squares), 20 (white squares), 30 (black circles), 40 (white circles), 50 (black triangles), 60 (white triangles), or 70 nM (gray circles) IN. MgCl2 concentration was 10 mM. B, 3'-processing kinetics in conditions of increasing ionic strength: 30 mM (black circles), 35 mM (white squares), or 40 mM MgCl2 (black squares). Concentrations of ODN and IN were 4 nM and 150 nM, respectively. Activities in A and B were calculated using rSDS. C, 3'-processing activity at t (Actmax) as a function of initial fractional saturation function. Actmax corresponds to the maximal fraction of released dinucleotides for given conditions of IN:DNA ratio or MgCl2 concentration (plateau in A and B) and was estimated by fitting: Act = Actmax x (1 – e–kobsxt). Y was calculated using rmax = 0.226. D, specific activity (Â = activity/Y) as a function of the IN:DNA ratio. Total activity was normalized using fractional saturation function (Y), giving 3'-processing activity per complex.

View this table: [in this window] [in a new window]   TABLE 2 Correlation times (37 °C) obtained for IN·ODN complexes using ds ODN HIV-a5F

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Kinetic Analysis of 3'-Processing Activity by the Anisotropy-based Method—The real-time assay described here is based on the use of a DNA duplex fluorescein-labeled on the terminal-3' GT. It allows quantification of the cleavage reaction resulting in physical separation of the processed 19/21-mer DNA (first product) and the fluorescently labeled dinucleotide (second product). IN assays based on fluorescence intensity (fluorescence resonance energy transfer or fluorescence quenching) have been described in previous studies (32–35). Unlike electrophoresis-based methods, which can be used to analyze discrete time points only, fluorescence methods present the significant advantage of allowing continuous monitoring of the reaction for kinetic studies of IN in the same sample. However, only the anisotropy-based method can be used to study both the DNA-binding step and subsequent activity in the same assay. Using various DNA sequences, we showed that IN does not demonstrate highly selective binding to the viral sequence over other sequences, but, in contrast, displayed strong selectivity for catalysis (Fig. 3). This result is not subject to possible bias due to indirect comparison, as might be the case when DNA binding and catalysis are studied by different methods. It suggests that IN first binds viral DNA in a nonspecific manner and that subsequent relaxation leads to the formation of a specific and catalytically competent complex before catalysis. This relaxation step must be very slow to account for the low single-turnover rate constant as previously reported (25).

View larger version (11K): [in this window] [in a new window]   FIGURE 7. Apparent Kd values of IN as a function of DNA length (number of nucleotides). Values of Kd,app were determined for ss (white squares) or ds ODNs (black squares) as indicated under "Experimental Procedures."

View larger version (30K): [in this window] [in a new window]   FIGURE 8. Correlation time distributions of the IN·DNA complexes obtained at different IN:DNA ratios. Representative samples corresponding to different r values in the decreasing phase of Fig. 5A were studied by TFA at 37 °C. IN:DNA ratios were 40:1 (A), 67:1 (B), 100:1 (C), 200:1 (D), and 400:1 (E). The different samples displayed similar lifetime distributions: 1 = 0.11 ± 0.06 ns (26%); 2 = 0.4 ± 0.1 ns (19%); 3 = 1.9 ± 0.1 ns (14%); 4 = 3.6 ± 0.2 ns (41%). Complete data ( and i/0) are reported in Table 2.

Our conclusion that the dimeric form of IN is active for 3'-processing under native conditions strongly parallels those obtained by Parissi and coworkers (41) with purified cross-linked multimers. The aggregation of IN on DNA was found to be strongly dependent on the IN:DNA ratio and was detrimental for 3'-processing, as previously reported for concerted integration (42). The bell-shaped curve obtained in Fig. 5 most likely originates from competition between the cooperative binding of IN on DNA leading to a dimeric form (specific IN-IN interaction, which is favored by increasing the IN:DNA ratio and enhances 3'-processing activity) and aggregation (nonspecific interactions also favored by increasing the IN:DNA ratio but detrimental for 3'-processing). The bell-shaped behavior is also compatible with the results of a recent cross-linking study (41) suggesting that monomers and tetramers display neither 3'-processing or half-strand transfer (transfer of a single viral DNA end) activity, whereas dimers are competent for both these reactions, and only tetramers catalyze full-site concerted integration. Our results suggest that, in vitro, IN aggregates do not much hinder 3'-processing under IN:DNA ratio conditions leading to the formation of dimers, but that such aggregates may impede tetrameric organization in a highly competitive manner. However, TFA experiments did not indicate whether the tetrameric form on one LTR end actually existed, because the intermediate distributions (Fig. 8, B–D) probably corresponded to mixtures of different multimeric forms (polydisperse solutions). It is therefore difficult to assess 3'-processing activity of tetramers, because IN:DNA ratios shifting dimer-tetramer equilibrium toward tetramers also gave rise to IN aggregation. Although recombinant IN alone is sufficient for concerted integration in vitro (41–44), this reaction remains much less efficient with recombinant IN than with the purified pre-integration complex (45) or virion-derived IN (46). The main limiting factor may be the multimeric organization of IN to yield the correct tetrameric form on DNA compatible with full integration process: aggregation is probably more limiting for DNA integration, which requires tetramers, than for 3'-processing, which requires dimers. Moreover, samples containing many aggregated forms displayed significant 3'-processing activity (one-third that of dimeric IN). The reason for this residual activity remains unclear.

Li and Craigie (44) recently showed that blunt-ended DNAs are more efficient substrates for concerted integration than preprocessed DNAs, whereas half-integration is more efficient with preprocessed DNA. These data, together with our results and those of Parissi et al. (41), suggest that dimers of IN are necessary and sufficient for 3'-processing and half-integration and that the 3'-processing step probably promotes a conformational change of IN compatible with concerted integration. This conformational change may play a key role in promoting the IN tetramerization required for concerted integration. IN is known to exist in a monomer-dimer-tetramer equilibrium in solution (16, 17, 24, 38). The multimeric form is modified on DNA, and the final quaternary structure depends on the IN:DNA ratio and probably also on the nature of the DNA substrate. From this point of view, the tetrameric organization of IN (a dimer of dimers) may be stabilized by the simultaneous presence of two LTR ends (47) and it highly suggested that the condensation-distortion properties of DNA play a key role in bringing the two ends of the viral DNA close enough in space for a dimer of dimers to form. A dimer at each LTR end, as described here, is consistent with current models based on cross-linking (48) or molecular dynamics data (49), and the 5-bp separation at target level is compatible with a tetrameric complex rather than a dimer. Several models of the tetrameric active complex exist for concerted integration: the 5-bp separation is directly compatible with a tetrameric complex displaying 2-fold symmetry, as modeled by McCammon and coworkers (31) or a tetrameric complex assuming target DNA bending (48, 50) or a hinging motion of the dimer interface (7). The formation of a dimer of dimers is probably mediated by the C-terminal domain, as suggested by the x-ray structure of the catalytic core/C-terminal double domain (9). Moreover, residue Trp-235 in the C-terminal domain has a significant effect on concerted integration but not on half-integration (44). The resulting symmetric organization, dispensable for 3'-processing but not for integration, may be favored or stabilized by the symmetric base sequence of the target DNA. Such symmetry in integration sites has recently been reported (51, 52).

View larger version (16K): [in this window] [in a new window]   FIGURE 9. Model of the 3'-processing reaction according to binding/multimerization and catalytic parameters. Monomers (M) preferentially bind to DNA in a cooperative manner and form a dimer (D) on the LTR at one end of the DNA (see text). This model involves the conversion of large multimers (INn 4) into smaller species prior to or simultaneously with DNA binding. Once the IN2·DNA complex has been formed, a slow D D* relaxation step, generating a catalytically competent complex, accounts for the very low single-turnover rate constant (kchemistry).

	FOOTNOTES

 
^* This work was supported by grants from the TrioH European Project (FP6 Grant 503480), the CNRS, the French National Agency for Research against AIDS, and the Russian Foundation for Basic Research (Grants 04-04-22000 and 05-04-48743). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 33-147-40-23-94; Fax: 33-147-40-76-84; E-mail: deprez{at}lbpa.ens-cachan.fr.

² The abbreviations used are: HIV, human immunodeficiency virus; IN, integrase; LTR, long terminal repeat; ODN, oligodeoxynucleotide; ds, double-stranded; ss, single-stranded; r, steady-state fluorescence anisotropy; TFA, time-resolved fluorescence anisotropy; TAMRA, carboxytetramethylrhodamine; wt, wild-type.

	ACKNOWLEDGMENTS

 
We thank Dr. H. Leh for helpful discussions and G. Mbemba for technical assistance.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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