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J. Biol. Chem., Vol. 281, Issue 17, 11530-11540, April 28, 2006
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1
From the
Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119992 Moscow, Russia and LBPA, UMR 8113 CNRS, Ecole Normale Supérieure de Cachan, 61 Avenue du Président Wilson, 94235 Cachan Cedex, France
Received for publication, November 15, 2005 , and in revised form, February 21, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Strand transfer and 3'-end processing reactions may be carried out in vitro, using recombinant HIV IN, DNA duplexes mimicking U3 or U5 sequences of LTRs, and divalent metal ions, such as Mg2+ or Mn2+. However, the Mn2+- and Mg2+-dependent activities of IN are not equivalent, with lower specificity reported for Mn2+-dependent IN (1, 2). Moreover, the inhibition of HIV-1 IN by compounds such as 
-diketo acids, which interact with the active site of HIV-1 IN, is also metal-dependent (3). The metal ion is coordinated within the IN catalytic core by two Asp residues (Asp-64 and Asp-116) and water molecules (4–6). These Asp residues are part of the D,D(35)E motif, a triad of invariant acidic amino acids found in all retroviral integrases (7) and ultimately required for catalytic activity (2, 8–10). Binding of the metal cofactor by the amino acids of the D,D(35)E motif causes both local and long range conformational changes and the activation of IN (5, 11). The IN catalytic core binds both the metal cofactor and the substrate. These conformational changes therefore influence the specificity of IN interactions with substrate DNA. Differences in the coordination properties of Mg2+ and Mn2+ions may result in different conformational changes in the IN catalytic core. This may account for differences in the specificity of HIV-1 IN interactions with substrate DNA (1, 2), inhibitors (3, 12), and the nucleophiles used to carry out 3'-end processing (13). As IN is more sensitive to the structure of molecules interacting with its catalytic core in the presence of magnesium ions, it has been suggested that the catalytic core of this molecule is more rigid in the presence of magnesium ions, as reported for other magnesium-dependent enzymes (1).
IN has three domains: the N-terminal, catalytic, and C-terminal domains. The structure of each domain, and of two-domain fragments, has been determined by x-ray crystallography or NMR spectroscopy (4, 6, 14–16). However, neither the structure of the entire enzyme nor that of the IN-viral DNA complex has yet been determined. Cross-linking experiments have been used to study contacts between IN and viral DNA, and it has been found that the catalytic core (residues 50–212) and C-terminal domain (residues 213–288) are the principal areas involved in DNA binding. It has been shown that the Lys-159 residue of HIV-1 integrase may interact with the N-7 position of the third adenosine of the processed viral DNA strand (17) and that Glu-152 may also interact with this adenosine (18). Gln-148 and Tyr-143 have been shown to bind to the terminal nucleotides of the processed strands of viral DNA, and the thymidine at position 7 interacts with amino acids from the C-terminal domain (1). Glu-246 is also thought to be located near the C-2' atom of the 2'-deoxyadenosine at position 7 in the unprocessed strand (19). Nevertheless, the protein/DNA interactions through which IN forms a stable and catalytically active complex with viral DNA are not yet completely understood.
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We therefore investigated the effect of the double-helix structure of viral DNA on HIV-1 integrase activity in the 3'-end processing reaction, as a means of identifying the structural features of the viral DNA determining the sequence specificity of its recognition and processing by IN. We synthesized a set of modified DNA duplexes, with sequences mimicking the U5 sequence of LTR of the viral DNA (U5 substrate). Nucleosides at various positions in the processed strand, the unprocessed strand, or both were replaced by a non-nucleoside insertion, 1,3-propanediol, or nucleosides containing modified sugar residues, 2'-aminonucleosides and 2'-O-methylnucleosides (Fig. 1). Because the conserved CA sequence adenosine is considered crucial for 3'-end processing and strand transfer activities, we also prepared a number of U5 substrate analogs in which the A/T pair was replaced by unpaired nucleotides in the 3rd position and a 2,6-diaminopurine replaced the conserved adenine.
We have shown that IN probably recognizes the viral DNA by forming at least two hydrogen bonds with the adenine at position 3. The complementary base, thymine, does not interact with IN. We suggest that IN recognizes the fine structure of the sugar-phosphate backbone rather than heterocyclic bases at other positions. We also found that interactions with the unprocessed strand were more important for specific substrate recognition by IN than interactions with the processed strand. We showed that local destabilization of the A/T pair at position 3 was required for 3'-end processing, allowing such processing to occur even at internal positions of the U5 substrate within DNA. Finally, we studied IN/DNA interactions in the presence of Mn2+ and Mg2+ ions, investigating the differences in substrate recognition specificity in the presence of these two cofactors. We found that modification of the nucleosides in the 6th, 5th, and especially the 3rd positions influenced 3'-end processing more strongly in the presence of Mg2+ than presence of Mn2+ in the ions, whereas modification of base pairs 7–9 affected 3'-end processing equally in the presence of both ions. We suggest that positions 3–6 within the substrate DNA interact with the IN catalytic core that binds the metal cofactor.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-cyanoethyl phosphoramidite procedure. The resulting oligonucleotides were purified by electrophoresis in a 20% denaturing polyacrylamide gel. Oligonucleotides containing 2'-(3-aminopropionyl)amino-2'-deoxyarabinoadenosine (Fig. 1C) and 2'-amino-2'-deoxyuridine (Fig. 1B) were synthesized as described previously (27, 28). Oligonucleotides containing 1,3-propanediol insertions (Fig. 1A) were synthesized using the commercially available Spacer C3 (Glen Research). An oligonucleotide containing 2,6-diaminopurine was synthesized using commercially available 2-amino-dA-CE phosphoramidite (Glen Research). Structures of all modified oligonucleotides were confirmed by matrix-assisted laser desorption/ionization time of flight. Hybridization of oligonucleotides U5B and U5A (U5B being the processed strand) gave the wild-type U5 integrase substrate. The fluorescein-labeled oligonucleotides U5B-fl (5'-fl-GTGTGGAAAATCTCTAGCAGT-3') and U5A-fl (3'-fl-CACACCTTTTAGAGATCGTCA-5') used for IN/DNA binding studies were synthesized as follows: oligonucleotide U5B, with an amino group at its 5'-end, and oligonucleotide U5A, with an amino group at its 3'-end, were synthesized with 5'-amino-modifier C6 reagent and 3'-amino-modifier C3 controlled pore glass (Glen Research), respectively. The resulting oligonucleotides were dissolved in a mixture of 60 µl of H2O, 20 µl of 1 M carbonate buffer (pH 11), and 80 µl of dimethylformamide. We then added 1 mg of fluorescein isothiocyanate to each solution. The mixtures were incubated in the dark at room temperature for 4–12 h, and 1 ml of 2% LiClO4 in acetone was added to precipitate the oligonucleotides. The reaction mixtures were analyzed by reversed-phase high performance liquid chromatography in ion pair mode, and the labeling efficiency was found to be greater than 90%. The conjugates were purified by electrophoresis in a 20% polyacrylamide denaturing gel, and the presence of fluorescein was confirmed by absorption spectroscopy.
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Integrase—Detergent-free recombinant integrase protein was produced in Escherichia coli and purified as described previously (29).
Thermal Denaturation Studies—Oligonucleotide duplexes (1 µM in 20 mM Tris-HCl buffer (pH 7.0), 7.5 mM MgCl2) were heated at 90 °C for 3 min and slowly cooled to 10 °C. Melting studies were carried out in Teflon-stoppered 1-cm path length quartz cells, using a Hitachi 100–20 spectrophotometer equipped with a thermal programmer. We monitored absorbance at 260 nm as the temperature was increased at a rate of 0.5 °C/min. Melting temperatures (Tm) were determined by fitting the first derivative of absorbance with respect to 1/T. Uncertainty in Tm was estimated to a precision of ±0.5 °C by repeating experiments.
Preparation of 5'-Radiolabeled DNA Duplexes—For binding and processing assays, 10 pmol of the oligonucleotide U5B, or one of its modified analogs, was labeled using T4 polynucleotide kinase and 50 µCi of [
-32P]ATP (3000 Ci/mmol). T4 polynucleotide kinase was then inactivated by adding EDTA and heating at 65 °C for 5 min. The corresponding complementary unlabeled oligonucleotide, U5A, or its modified analog (10 pmol) was then added to each of the labeled oligonucleotides. The mixtures were heated at 90 °C for 3 min, and the DNA duplexes were allowed to anneal by slowly cooling to room temperature. DNA duplexes were purified using Micro Bio-Spin columns P-6 (Bio-Rad).
3'-End Processing Assay—The 3'-end processing reaction was carried out in a buffer containing 20 mM HEPES (pH 7.2), 1 mM DTT, and 7.5 mM MnCl2 or MgCl2 using the IN substrate U5B/U5A or an analog containing modifications in one or both strands. 32P-Labeled DNA substrates (1.25 nM) were incubated in 20 µl of the buffer with 100 nM integrase at 37 °C. Aliquots were taken after 5, 10, 20, 30, 60, and 120 min. The reaction was stopped by adding 80 µl of a stop solution (7 mM EDTA, 0.3 M sodium acetate, 10 mM Tris-HCl (pH 8)). IN was extracted in phenol/chloroform, and DNA fragments were precipitated with ethanol. The products were suspended in loading buffer (80% formamide, 0.05% bromphenol blue, 0.05% xylene cyanol) and separated on 20% polyacrylamide denaturing gels. Gels were analyzed on a Storm 840TM PhosphorImager (Amersham Biosciences) and quantified with Image-QuantTM 4.1 software (Fig. 3). We fitted a nonlinear regression curve to the experimental data, and initial rates were calculated as the tangent to the kinetic curves for t = 0. Rate data for modified substrates were normalized to the initial rate of 3'-end processing of the natural U5 substrate. All 3'-end processing experiments were carried out at least three times; the standard deviation was between 5 and 10%.
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Steady-state Fluorescence Anisotropy Measurements—Steady-state fluorescence anisotropy values were recorded on a Beacon 2000 apparatus (Panvera, Madison, WI) in a thermostated cell, at 37 °C for kinetics studies or 20 °C for competition experiments. For kinetics studies, fluorescein-labeled DNA duplexes (4 nM) (wild-type substrate or modified oligonucleotides) were added to a 200-µl buffered solution containing 20 mM HEPES (pH 7.2), 1 mM DTT, and 7.5 mM MgCl2. IN-DNA complex formation was then triggered by adding IN (200 nM final concentration). A constant level of 20 mM NaCl was maintained in all reaction mixtures. Fluorescence anisotropy values were then recorded, and the binding curves were used to determine the pseudo first-order binding rate constant, kon = k1 x [IN] (where k1 is the second-order binding rate constant). A single-exponential model was fitted to the data for DNA-IN complex formation.
For competition experiments, fluorescein-labeled DNA duplexes (2 nM) were first incubated with various concentrations of unlabeled DNA duplexes (either unmodified or modified oligonucleotides) in a buffered solution containing 20 mM HEPES (pH 7.2), 1 mM DTT, 20 mM NaCl, and 5 mM MgCl2. IN (120 nM final concentration) was then added, and steady-state anisotropy values (r) were recorded after equilibrium was reached. The r value corresponding to free fluorescein-labeled DNA was subtracted from the measured r value to give 
r. Finally, r values were plotted against competitor concentration to determine apparent Kd (concentration of unlabeled DNA decreasing the initial r value by 50%).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Modifications at Position 12—We investigated the influence of U5 substrate modifications on the ability of IN to mediate 3'-end processing. We found that replacement of the nucleosides at position 12 by the non-nucleoside insertion P did not affect IN activity in presence of Mg2+ or Mn2+ ions (supplemental Table S2). These nucleosides therefore do not form contacts with IN that are important for catalytic activity. By contrast, modifications at other sites on the U5 substrate significantly influenced the processing rate.
Modifications at Positions 7–9—Both non-nucleoside insertions and nucleoside modifications at position 9 decreased the 3'-end processing rate (Table 1). Similar effects were observed for modifications of the processed and unprocessed strands. Non-nucleoside insertions decreased the reaction rate more strongly than the insertion of a modified nucleoside. The 3'-end processing rate was lower if both substrate strands were modified. Therefore, nucleosides at position 9 in both strands of the DNA are involved in interactions with IN.
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It should be especially noted that replacing position 7 of the U5B strand with 2'-aminouridine increased 3'-end processing rate (Table 1). This can be explained by the formation of an additional contact between the amino group of 2'-aminouridine and an amino acid inside IN. It may be an extra hydrogen bond or an electrostatic interaction that occurs if this amino group is at least partially protonated (pKa = 6.2 (33, 34)). We verified this by treating an oligonucleotide containing 2'-aminouridine at position 7 (B-N7) with succinic anhydride, to give an oligonucleotide containing a carboxyl group (B-N7-ac) (Fig. 2A). We found that the 3'-processing rates of the duplex with this carboxyl group (B-N7-ac/U5A) and nonmodified U5 substrate were the same (Fig. 2B). Therefore, the increase in the 3'-processing rate of the substrate containing 2'-aminonucleoside at the processed strand position 7 is because of the presence of the 2'-amino group. This processing rate increase was also observed when the seventh nucleosides in both strands were modified. However, the effect was less, probably because of the opposition of two different effects: an increase in processing rate caused by the modification of the processed strand, and a decrease caused by the modification of the nonprocessed strand. Finally, for all modifications at positions 7–9, an equal effect on processing rate was observed in the presence of either Mn2+ or Mg2+ ions (Table 1).
Modifications of Positions 5 and 6—By contrast with the positions 7 and 8, modifications of the nucleosides in either strand at positions 5 and 6 affected the 3'-processing rate (Table 2). However, the rate decrease was much greater when the nonprocessed strand was modified. Replacement of native nucleosides by both the non-nucleoside insertion (P) and the modified nucleosides, 2'-aminonucleosides or 2'-O-methylnucleosides, resulted in a similar effect (Table 2). Therefore, IN was sensitive to any distortion of the double helix in this DNA region and not especially affected by heterocyclic base removing. The inhibition effect of the nucleoside modification was much greater in the presence presence of Mg2+ than in the Mn2+ ions (Table 2).
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Replacement of the thymidine at position 3 in the unprocessed strand by 1,3-propanediol (P) or any modified nucleoside (UN or UM) increased the 3'-end processing rate (Table 3 and Fig. 3), regardless of the cofactor used. All these modifications at position 3 destabilized the DNA double helix (supplemental Table S1). This destabilization may stimulate 3'-end processing, consistent with previous data demonstrating an increase in 3'-processing activity because of destabilization of the 3rd bp (26, 35, 36).
The duplex with non-nucleoside insertions in both strands was not processed, regardless of the cofactor used (Table 3). In the presence of Mg2+ ions, no 3'-processing was also observed for the duplex containing 2'-aminonucleosides in both strands, because of the inhibitory effect of the processed strand modification (Fig. 3 and Table 3). As replacing the native A by aAN did not affect 3'-end processing in the presence of Mn2+ ions, we observed an increase in reaction rate for the substrate analog containing two 2'-aminonucleosides in presence of this ion (Table 3). This effect may be due to the modification of both strands, further destabilizing the duplex.
As destabilization of the A/T base pair at position 3 stimulated 3'-end processing, we hypothesized that the stabilization of this base pair would decrease the processing rate. We tested this hypothesis by studying 3'-end processing of U5 substrate analogs containing 2,6-diaminopurine (DAP) instead of the adenine at position 3 of the U5B strand (Fig. 5A). The DAP base stabilizes the DNA structure by forming three hydrogen bonds with the complementary thymine, instead of two bonds for the normal Watson-Crick pair A/T (Fig. 5B) (37). Processing was strongly inhibited in the presence of either metallic cofactor following the incorporation of DAP into the U5B strand (duplex B-DAP/U5A) (Fig. 5, C and D). However, the 3'-end processing rate was increased by elimination of the hydrogen bonding between DAP and T because of replacement of the T residue by 1,3-propanediol (duplex B-DAP/A-P3). Processing was as rapid as for the wild-type substrate in the presence of Mn2+ ions (Fig. 5C). A smaller increase in processing rate was observed in presence of Mg2+ ions (Fig. 5D).
We also studied interactions between IN and two 26-mer duplexes, because we assumed that A/T pair destabilization would enable IN to mediate cleavage of the U5 sequence in a more extended DNA duplex (Fig. 6). One duplex contained the sequence of the U5 substrate with 5 additional base pairs (26B/A). In the second duplex (26B/Amut), we replaced the thymine preceding the conserved adenine by an adenine, resulting in the noncomplementary pair A/A (Fig. 6A). The 3'-end processing efficiency of the 26B/A duplex was very low in the presence of Mn2+ ions (Fig. 6B), and no processing was observed in the presence of Mg2+ ions (Fig. 6C). In the case of the duplex containing the noncomplementary pair (26B/Amut), the 3' -processing efficiency increased in the presence of Mn2+ ions (Fig. 6B). Moreover, IN was able to cleave the mutated substrate even in presence of Mg2+ ions (Fig. 6C). Thus, A/T pair destabilization is essential for DNA cleavage by IN.
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We then investigated IN/DNA interactions by steady-state fluorescence anisotropy (Table 4). The fluorescein-labeled wild-type DNA substrate was incubated in the presence of various concentrations of unlabeled modified DNA duplexes (Table 4) before the addition of IN. We calculated apparent Kd values from the competition curves for the modified DNA duplexes (Table 4). Consistent with the results of electrophoretic mobility shift assays, the apparent affinities of IN for the various modified duplexes were comparable with the one for wild-type substrate. A slight affinity increase was obtained for substrates containing the abasic site, which display no activity under magnesium conditions (U5B/A-P5 and B-P3/U5A K'd 
8.5 nM, as compared with U5B/U5A Kd' 21 nM). Moreover, other DNA substrates (U5B/A-N6 and B-N3/U5A) that display no 3'-processing activity have an apparent affinity for IN comparable with the wild-type substrate. Finally, processing of the modified duplex B-N7/U5A, which is characterized by the weakest affinity (Kd' 36 nM), was stimulated (Table 1). Therefore, no correlation between the apparent affinity and the specific activity was detected.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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It is important that all our modifications introduced a local distortion in the DNA double helix structure, decreasing Tm (supplemental Table S1). The instability caused by 2'-amino modification probably results from heterocyclic base destacking (27), causing base displacement in the double helix structure. In the case of UM, the destabilization may result from its C-3'-endo sugar puckering typical of the A-form geometry of RNA (32, 40). However, 2'-modified nucleosides retain their heterocyclic bases and may maintain at least some contacts with IN, whereas the incorporation of 1,3-propanediol results in the complete elimination of all functional groups that might interact with IN.
Most of the substrate positions involved in the specific activity of IN are located upstream from the 9th bp (20, 22, 25). However, nucleotides at positions 10–13 have also been shown to be important (1). We found that modifications at position 12 did not alter IN activity. We therefore focused on positions 3–9. We found that their modifications altered the 3'-end processing rate. As the conflicting results reported in previous publications may arise from the differential use of Mn2+ or Mg2+ ions, we also compared, for each modification, the 3'-end processing rate in the presence of these two cations.
We found that the catalytic step was sensitive to modifications to DNA-substrate structure but that the modifications had no effect on substrate binding to IN. In our hands, it was not possible to distinguish between specific and unspecific binding of IN to DNA. The Kd value derived from binding to the U5 sequence was very close to the one obtained with nonspecific double-stranded DNA (25 nM; data not shown). Under our enzymatic conditions (E > S), IN activity was correlated to the amount of IN-DNA complexes. It was therefore crucial to verify whether the substrate modifications did not alter the quantity of complexes formed in vitro. No modification of IN binding was observed, thus indicating that the alteration of the catalysis was related to a post-binding event, most likely a modification of the DNA conformation, which prevented the cleavage to occur.
Integrase Interacts with the DNA Sugar-Phosphate Backbone at Positions 5–9—We identified three different sites, between nucleosides 5 and 9 inclusive, where modification affected 3'-end processing rate in different ways.
All modifications at position 9 affected processing rate, but this effect was moderate in all cases (Table 1). The strongest effect was obtained by replacing the base pair by 1,3-propanediol (P). However, despite the P insertion causing the largest DNA distortion (the Tm shift was 8.3 °C) because of the disruption of both hydrogen bonding and heterocyclic base stacking, the processing rate was no more than halved. This suggests that IN interacts with the sugar-phosphate backbone, without making specific contacts with the bases. Moreover, single modifications on either strand affected IN activity equally, suggesting that this interaction likely involves both strands.
At positions 7 and 8, the total removal of heterocyclic bases from the processed strand had no significant effect on 3'-end processing rate (Table 1, modification P), ruling out the possibility of specific contact with heterocyclic bases. By contrast, similar modifications of the unprocessed strand decreased IN activity (Table 1, modification P) thus allowing specific interactions with IN for heterocyclic bases from this strand. It has been shown, however, that substitutions at positions 7 and 8 with other complementary base pairs, which do not disturb the DNA double helix structure but change the pattern of putative bases contacts with IN, do not affect 3'-end processing (1). Thus, the inhibitory effect of P insertion results from the loss of IN contacts with the sugar-phosphate backbone because of its local distortion rather than a loss of contacts with heterocyclic bases.
Any modification of the fifth and sixth native nucleosides, either by P insertion or by replacement with 2'-modified nucleosides, led to a large decrease in the 3'-end processing rate (Table 2). Similar levels of inhibition were observed with all substrate modifications at position 6, suggesting that IN recognizes the conformation of the DNA backbone rather than nucleoside bases. Previous studies have shown that replacing the guanine at position 5 with O6-methylguanine decreases substrate cleavage (26). It has been suggested that IN interacts with this heterocyclic base via the major groove of the DNA. However, the 5th and 6th bp are inverted in the U5 and U3 extremities (5'-A(G/C)T-3' in U5 and 5'-T(C/G)A-3' in U3), although both sequences are substrates of the enzyme. This observation is not consistent with the direct recognition of the bases at these positions by IN. Moreover, the effect of O6-methylguanine may also result from the DNA structure distortion, because guanine O6-methylation modifies the conformation of the phosphodiester backbone at the modification site (41). It therefore seems more likely that IN recognizes a DNA fine structure generated by the G/C and A/T pairs at positions 5 and 6, respectively. Deformation of the double helix by either base replacement or nucleoside modification prevents DNA recognition, thereby strongly inhibiting 3'-end processing (Table 2).
We conclude that IN recognition involves only interactions with the DNA sugar-phosphate backbone at positions 5–9. Both nucleosides of the 9th bp participate in these interactions. For positions 5–8, modifications of the unprocessed strand decrease 3'-end processing rate more strongly than modifications of the processed strand (Tables 1 and 2), indicating that active IN-DNA complex formation primarily requires interactions with the unprocessed strand. Nevertheless, at positions 5 and 6, contact between IN and both strands is important for specific DNA recognition (Table 2).
Destabilization of the A/T Pair at Position 3 Is Necessary for 3'-End Processing—Modification of the thymidine at position 3 in the unprocessed strand, by P insertion or 2'-modified nucleosides (UN and UM), increased 3'-end processing rate (Table 3) probably because of local destabilization of the A/T base pair, as suggested previously (36). The processing stimulation following the thymidine replacement likely points to an absence of specific contact between IN and this base. Alternatively, one could consider that mispairing may result in a loss of a specific contact otherwise required for the unpairing step. However it must be noted that according to Ref. 42, binding of the avian homolog of HIV integrase to its DNA substrate enhances the accessibility and the subsequent modification of the third thymidine by KMnO4. Thus, although integrase disrupts the A/T base pair, it does not protect the heterocyclic base from the modification, thereby ruling out a close contact between the base and the enzyme. The stimulation caused by A/T base pair destabilization is also consistent with the inhibition elicited by replacing the adenine at position 3 with DAP, a base known to stabilize the double helix as a result of the formation of three hydrogen bonds (Fig. 5B) (37). Accordingly, the disruption of these hydrogen bonds by replacing thymidine in the DAP/T base pair by 1,3-propanediol (duplex B-DAP/A-P3) led to an increase in 3'-end processing rate (Fig. 5, C and D).
The negative effect of DAP substitution on 3'-processing has been reported before, and it has been suggested that DAP disrupts a specific contact between IN and the C-2 position of the adenine at position 3 (26). However, such a contact is not consistent with the efficient cleavage of the mismatched substrate B-3T/U5A (Fig. 4) or the stimulation observed if the thymidine complementary to DAP is replaced by 1,3-propanediol (Fig. 5C). We therefore suggest that DAP prevents the local A/T pair destabilization that is essential for efficient processing. The necessity of this destabilization also found for avian sarcoma virus integrase (42) may account for the strict conservation of the CA/TG sequence in integrase substrates, as intrinsic conformational mobility is known to favor DNA flexibility (43–47). Such a local destabilization allows IN activity, even at an internal position, as seen in the internal processing of a 26-mer duplex (26B/A). As predicted, processing efficiency increased considerably when the A/T base pair was replaced by a noncomplementary A/A pair (duplex 26B/Amut) (Fig. 6).
Specific and Metal-dependent Interactions with the Adenosine at Position 3 in the Processed Strand—It has been reported that replacement of the A/T base pair at position 3 by other complementary pairs (1, 35) or replacement of the adenine in this position with N 7-deaza-adenine (26) decreases 3'-end processing efficiency, suggesting that specific contacts with this adenine are required. However, other studies have shown that replacement of the A/T pair by either noncomplementary base pairs (35) or an abasic site (AP-site) (25) does not affect 3'-end processing efficiency. Our study reconciles these contradictory data by demonstrating the important role played by the metal cofactor. Indeed, we observed that the replacement of 2'-deoxyadenosine by P insertion completely inhibited 3'-processing in the presence of both Mg2+ and Mn2+ (Table 3). By contrast, the modification of this residue by 2'-aminonucleoside, aAN, yielded a metal-dependent effect; 3'-end processing was completely inhibited in the presence of Mg2+ ions, whereas no inhibition was observed in the presence of Mn2+ (Table 3). It has been suggested that Mg2+ coordination makes the active site of IN more rigid, whereas Mn2+ coordination does not (1, 4). The loss of flexibility caused by Mg2+ coordination may prevent IN from establishing specific contacts with the adenine within aAN, as the sugar modification changes the location of the adenine. As a result, 3'-processing of the aAN-containing substrate is inhibited in the presence of Mg2+ but unaffected in the presence of Mn2+, which maintains structural flexibility of IN.
We investigated possible specific contacts between IN and the adenine at position 3 by studying the processing of substrates in which this adenosine was replaced by other nucleosides (Fig. 4). Adenine, which is simultaneously involved in Watson-Crick base pairing, can form hydrogen bonds with IN via its N-7 atom (proton acceptor) and the exo-cyclic amino group (proton donor) in the major groove and via the N-3 atom (proton acceptor) in the minor groove. A specific contact between IN and the N-7 atom has been suggested, based on the decrease in processing efficiency by a factor of 3 caused by replacing the adenine at position 3 with N7-deaza-adenine, which cannot maintain this contact (26). The low processing rate observed for the C-containing duplex (B-3C/U5A) (Fig. 4B), for which a contact similar to that formed by N-7 is impossible, confirmed the necessity of this contact. However, the strong decrease in processing rate for the B-3G/U5A duplex (Fig. 4B), despite the presence of the same proton acceptors in guanine and adenine (N-3 and N-7 atoms), indicates that the NH2 group of adenine is also involved in an interaction with IN. The suggestion that two contacts are formed between the adenine and IN is supported by the finding that the T-containing duplex (B-3T/U5A) was processed almost as efficiently as the wild-type substrate (Fig. 4B). Indeed, thymine, which is not involved in Watson-Crick base pairing in this duplex, may form two hydrogen bonds with IN, involving O-4 as the proton acceptor (similar to N-7 of adenine) and the NH group as the proton donor. As the geometry of the bonds formed by thymine is not identical to that of the bonds formed by adenine, these bonds can be generated only if the structure of IN structure is flexible enough to accept T-containing duplexes as a substrate, i.e. in presence of Mn2+ ions. Clearly, these contacts cannot be formed if the thymine participates in base pairing, and as result, the 3'-end processing of a substrate analog containing the T/A pair instead of A/T is inefficient (35).
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Summary of IN/DNA Interactions—The interactions between IN and DNA substrate identified in this study are summarized in Fig. 8A. This scheme provides new insight into mechanistic similarities between IN and restriction endonucleases. Retroviral integrases and type II restriction enzymes have been reported to share conserved structural features (48). Thus, in the archetypal EcoRV/DNA interaction model, the protein contacts the base just upstream from the scissile bond but does not contact the complementary base in the opposite strand (Fig. 8B). Similarly, IN interacts with the adenine at position 3, but not its complementary thymine (Fig. 8A). Position –2 in the EcoRV recognition sequence, which is similar position 4 in the HIV-1 DNA, displays the highest level of contact within the EcoRV-DNA complex, with contacts established on both strands. It has been suggested that heterocyclic bases from the 4th bp interact with IN (1, 20, 21). Moreover, in addition to base-specific contacts, numerous contacts of EcoRV with the DNA backbone allow recognition of the cognate site through a sequence-dependent backbone conformation (49, 50). Regarding HIV-1 IN, it was found that specific protein mutations altering ionic interactions or hydrogen bonding result in a change of DNA recognition specificity when the mutated amino acids are located close to DNA positions 5–11 (51). These data support our conclusion that IN recognizes the fine conformation of DNA backbone at positions 5–9 rather than nucleoside bases.
Finally, EcoRV endonuclease initially binds DNA nonspecifically, subsequently forming a specific complex as a result of conformational changes in both the DNA and the enzyme (49). The DNA is locally distorted from a regular B form to a highly strained kinked conformation, leading to unwinding of the DNA, unstacking of the two central base pairs of the recognition site, and bending of the DNA (50). For IN, this bending and unstacking may be favored by the conformational mobility of the CA/TG fragment (43–44) and a loss of regular base stacking (45).
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains Tables S1 and S2 and Fig. S1.
1 To whom correspondence should be addressed: LBPA-CNRS UMR8113, Ecole Normale Supérieure de Cachan, 61 Avenue du Président Wilson, 94235 Cachan Cedex, France. Tel.: 33-1-47-40-76-75; Fax: 33-1-47-40-76-84; E-mail: mouscadet{at}lbpa.ens-cachan.fr.
2 The abbreviations used are: HIV-1, human immunodeficiency virus type 1; LTR, long terminal repeat; IN, integrase; DTT, dithiothreitol; DAP, 2,6-diaminopurine.
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ACKNOWLEDGMENTS |
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