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Supplemental Material can be found at: http://www.jbc.org/cgi/content/full/M605101200/DC1 THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 43, pp. 32428 –32438, October 27, 2006 Printed in the U.S.A.

Effect of DNA Modifications on DNA Processing by HIV-1 Integrase and Inhibitor Binding ROLE OF DNA BACKBONE FLEXIBILITY AND AN OPEN CATALYTIC SITE *□ S

Received for publication, May 30, 2006, and in revised form, July 13, 2006 Published, JBC Papers in Press, August 30, 2006, DOI 10.1074/jbc.M605101200

Allison A. Johnson‡, Jane M. Sayer§, Haruhiko Yagi§, Sachindra S. Patil¶, Franc¸oise Debart储, Martin A. Maier**, David R. Corey‡‡, Jean-Jacques Vasseur储, Terrence R. Burke, Jr.¶, Victor E. Marquez¶, Donald M. Jerina§, and Yves Pommier‡1 From the ‡Laboratory of Molecular Pharmacology, Center for Cancer Research, NCI, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892, §Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892, ¶Laboratory of Medicinal Chemistry, Center for Cancer Research, NCI-Frederick, National Institutes of Health, Department of Health and Human Services, Frederick, Maryland 20892, 储LCOBS UMR 5625 CNRS-Universite´ Montpellier II, 34095 Montpellier, France, **Isis Pharmaceuticals, Inc., Carlsbad, California 92008, and ‡‡Departments of Pharmacology and Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75235

* This work was supported by the Intramural Research Program of the NCI, Center for Cancer Research, and of the NIDDK, National Institutes of Health, National Institutes of Health Grant GM60642 (to D. R. C.), and CNRS (to J. J. V. and F. D.). 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. □ S The on-line version of this article (available at http://www.jbc.org) contains additional text and supplemental Table 1. 1 To whom correspondence should be addressed: Laboratory of Molecular Pharmacology, Bldg. 37, Rm. 5068, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-496-5944; Fax: 301-402-0752; E-mail: [email protected].

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HIV-12 integrase (integrase) catalyzes insertion of cDNA copies of the viral genome into human chromosomes. Integrase binds to the ends (“att” sites) of each viral long terminal repeat (LTR) through sequence-specific recognition of a conserved 5⬘-CA within the sequence 5⬘-GCAGT. In the first of two reactions, integrase cleaves the 3⬘-ends of the viral DNA, releasing the terminal 5⬘-GT dinucleotide (3⬘-processing, 3⬘-P). In the second reaction, the free 3⬘-hydroxyl of the conserved adenine provides the nucleophile for insertion of the viral cDNA into a chromosome (strand transfer, ST). Gap repair and ligation between the viral and cellular DNA are performed by cellular factors. (For recent reviews and insights on integration, see Refs. 1–5.) Determination of the molecular interactions between integrase and its DNA substrates (viral and chromosomal DNA) has proven challenging, and a co-crystal of these components remains elusive. Biochemical studies have revealed contact points between the viral DNA and integrase. Integrase has an absolute requirement for the conserved 5⬘-CA adjoining the 3⬘-P site (underlined in Fig. 1A). The efficiency of 3⬘-P is also dramatically decreased by changes to the G immediately 5⬘ to the conserved CA dinucleotide (6 – 8). The conserved adenine, substituted by 5-iododeoxyuracil as a photocross-linker, forms a photocross-link to Lys-159 of integrase (9). Residue Lys-159 also contacts the phosphate 5⬘ to the conserved deoxyadenosine (10). Mutagenesis showed that Tyr-143 and probably Gln-148 interact with the 5⬘-overhang resulting from 3⬘-P (9, 11). Moreover, disulfide cross-linking revealed proximity of the integrase amino acid residue 148 (Q148C mutant) to the second (cytosine) base and of residue 246 (E246C mutant) to the seventh (adenine) base from the 5⬘-end of the lower strand of the U5 LTR (see Fig. 1A) (12, 13). Interactions between the backbone of the viral DNA and 2

The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; 3⬘-P, 3⬘-processing; BaP DE, benzo[a]pyrene 7,8-diol 9,10-epoxide; DKA, diketo acid; LNA, locked nucleic acid; LTR, long terminal repeat; Ma-DKA, monoazido diketo acid; ST, strand transfer; MOPS, 4-morpholinepropanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

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Integration of the viral cDNA into host chromosomes is required for viral replication. Human immunodeficiency virus integrase catalyzes two sequential reactions, 3ⴕ-processing (3ⴕ-P) and strand transfer (ST). The first integrase inhibitors are undergoing clinical trial, but interactions of inhibitors with integrase and DNA are not well understood in the absence of a co-crystal structure. To increase our understanding of integrase interactions with DNA, we examined integrase catalysis with oligonucleotides containing DNA backbone, base, and groove modifications placed at unique positions surrounding the 3ⴕ-processing site. 3ⴕ-Processing was blocked with substrates containing constrained sugars and ␣-anomeric residues, suggesting that integrase requires flexibility of the phosphodiester backbone at the 3ⴕ-P site. Of several benzo[a]pyrene 7,8-diol 9,10-epoxide (BaP DE) adducts tested, only the adduct in the minor groove at the 3ⴕ-P site inhibited 3ⴕ-P, suggesting the importance of the minor groove contacts for 3ⴕ-P. ST occurred in the presence of bulky BaP DE DNA adducts attached to the end of the viral DNA suggesting opening of the active site for ST. Positionspecific effects of these BaP DE DNA adducts were found for inhibition of integrase by diketo acids. Together, these results demonstrate the importance of DNA structure and specific contacts with the viral DNA processing site for inhibition by integrase inhibitors.

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the ST site were important within the target DNA (14). Together, these observations suggest many stabilizing contacts between integrase, the tip of the viral cDNA LTR, and the target DNA. Additional information is required for higher resolution modeling and structure-based design of integrase inhibitors. Integrase 3⬘-P and ST can be examined with a simple in vitro assay using recombinant integrase, duplex oligonucleotides derived from the sequence of the last 21 bp of the U5 LTR (Fig. 1A), and a divalent metal cofactor. 3⬘-P produces a 19-mer product on denaturing sequencing gels as the 5⬘-GT dinucleotide is cleaved (see Fig. 1D, Ctl ⫹ IN lane). In the same assay, ST is achieved by insertion of the processed DNA into another identical duplex, resulting in a ladder of products migrating slower than the substrate DNA in denaturing sequencing gels. Here we present data from experiments using manganese so that our results are comparable with prior studies (7) and to create the most permissive conditions for integrase reactions. The data in Figs. 4 –7 were also performed with magnesium, with similar results (data not shown). The use of synthetic oligonucleotides allows studies of the effects on integrase activity of site-specifically placed DNA modifications. Here we focused on DNA backbone and base modifications surrounding the adenine of the conserved 5⬘-CA dinuFIGURE 1. Inhibition of HIV-1 integrase 3ⴕ-P by restriction of sugar puckering. A, sequence of the final 21 bp cleotide sequence to probe the DNA of the HIV-1 U5 long terminal repeat. The conserved 5⬘-CA is underlined, and the 3⬘-P site is indicated by a contacts between integrase and the triangle. B, normal nucleic acids exist in rapid equilibrium between north and south orientations of sugar viral DNA ends. We examined the puckering. The equilibrium tends toward the south for B-DNA and the north for RNA. C, the location (restricted nucleotide in boldface) and structures of the conformationally locked nucleosides are shown within the effect of several DNA backbone sequence of the five terminal nucleotides of the HIV LTR. Pymol images in stereo are derived from Protein Data modifications on integrase activity. Bank codes 1EK2, 1OF1, and 1I5W. D, representative gels showing the effect of north (n) and south (s) conformationally locked bicyclo[3.1.0]hexane nucleosides, LNA, and adenine ribonucleoside (riboA) at the site of 3⬘-P Oligonucleotides containing concompared with normal DNA (Ctl, control). The cleaved ribo-containing oligonucleotide migrated slower than formationally constrained sugars the fully deoxyribo-containing oligonucleotide, and the migration of the ribo-19-mer is noted. IN, integrase. attached to the conserved adenine were used to examine the conintegrase have been sparsely examined. Phosphate ethyla- formational preference for north (north bicyclo[3.1.0]hexane tion interference was used to locate phosphates that are crit- and locked nucleic acid [LNA]) or south (south bicical for integrase catalysis (14). Specific DNA backbone con- yclo[3.1.0]hexane) oriented sugars (Fig. 1C, see structures). A tacts required for ST near the insertion site were identified. On simple ribose substitution was also examined for comparison to the cleaved strand of the viral DNA, the phosphate 5⬘ to the LNA. Additionally, the effects of anomeric inversion on inteconserved adenine, as well as the two phosphates on the grase 3⬘-P and ST were examined using oligonucleotides concomplementary strand that are closest to the cleavage site, taining ␣-anomers around the 3⬘-P site (Fig. 2A). Substitution were important for ST. The two phosphates at and following of an ␣-anomer results in a change in the normal 5⬘–3⬘ direc-

Integrase Accesses Supple DNA Backbones from the Minor Groove

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MATERIALS AND METHODS Oligonucleotide Synthesis—All oligonucleotides were derived from the sequence of the last 21 bases of the HIV U5 LTR (Fig. 1A), with the exception of substitutions described. Unmodified, abasic site, and ribosecontaining oligonucleotides were commercially synthesized by IDT (Coralville, IA). 7-Deazaadenine and 2-aminopurine containing oligonucleotides were commercially synthesized by Midland Certified Reagent Co., Inc. (Midland, TX). Synthesis for bicyclo[3.1.0]hexane, LNA, ␣-anomer, and BaP-modified oligonucleotides is described in the Supplemental Material. FIGURE 2. Inhibition of HIV-1 integrase 3ⴕ-P by anomeric inversion. A, schematic structure of the last four All oligonucleotides were further nucleotides of the “␣1” oligonucleotide. The phosphate on the 3⬘-side of the conserved adenine (the scissile phosphate) is directly linked to the 3⬘-OH guanosine, resulting in polarity inversion. B, sequences of purified on denaturing 20% polyacoligonucleotides containing anomeric inversions. Oligonucleotide directionality is indicated by arrows rylamide gels. Single-stranded oliabove the sequences and ␣ notation at the site of polarity inversion. ␣1 has a 3⬘–3⬘ polarity inversion starting with the guanosine of the cleaved dinucleotide. ␣2 has a 3⬘–3⬘ polarity inversion starting with the gonucleotides were 5⬘-labeled using conserved adenosine. ␣3 has a polarity inversion only for the conserved adenosine (3⬘–3⬘ followed by T4 polynucleotide kinase (Invitro5⬘–5⬘ reversion). The 3⬘-P site is indicated by the triangle. C, representative gel showing the effect of ␣ gen) with [␥-32P]ATP (Amersham nucleotides on integrase (IN) 3⬘-P. Ctl indicates the normal DNA control. All oligonucleotides containing Biosciences) according to the mananomeric inversions are annealed to unmodified complementary strands. ufacturers’ instructions. Unincorporated nucleotide was removed tionality of DNA. The presence of an ␣-anomer in DNA does by mini Quickspin oligo column (Roche Applied Science). The not affect base pairing or duplex stability but can affect sugar duplex DNA was annealed by addition of an equal concentrapuckering (15). tion of the complementary strand, heating to 95 °C, and slow We also used covalent adducts derived from enantiomeric ben- cooling to room temperature. zo[a]pyrene 7,8-diol 9,10-epoxides (BaP DE) attached to either Integrase Reactions—Recombinant wild-type HIV-1 intesingle adenines or guanines to probe the position-specific effects of grase was purified from Escherichia coli as described (21) with bulky adducts in the major or minor groove of the viral DNA on the addition of 10% glycerol to all buffers. Integrase was incuintegrase 3⬘-P and ST. The hydrocarbon portion of the guanine bated with DNA substrates for 1 h at 37 °C. The reaction conN-2 adducts lies in the minor groove of the DNA and extends ditions were 500 nM integrase, 20 nM duplex DNA, 7.5 mM toward the 5⬘ or 3⬘ terminus of the adducted strand for the trans- MnCl , 5 mM NaCl, 14 mM 2-mercaptoethanol, and 20 mM 2 (S) and trans-(R) adducts, respectively, where S and R refer to the MOPS, pH 7.2. Reactions were quenched by the addition of an absolute configuration at the point of attachment of the 2-amino equal volume of gel loading dye (formamide containing 1% SDS, group of the hydrocarbon (see Fig. 3, D and E) (7, 16) We reported 0.25% bromphenol blue and xylene cyanol). Products were seppreviously that a BaP DE dG adduct in the minor groove of the 3⬘-P arated on 20% polyacrylamide denaturing sequencing gels. site blocked 3⬘-P (7). Here we examined the effect of dG minor Dried gels were visualized using a 445 SI PhosphorImager (Amergroove adducts attached to the 3⬘-processed dinucleotide, and we sham Biosciences). Densitometric analysis was performed using compared these results to those obtained with BaP DE adducts attached to the exocyclic N-6 amino group of adenines (Fig. 3, ImageQuant software from Amersham Biosciences. Schiff Base Cross-linking Assay—The Schiff base cross-linkA–C). These adenine adducts were used to probe the DNA major ing experiments were performed as described (22). Briefly, oligroove and DNA unwinding on enzyme/DNA interactions gonucleotides containing uracil at position U3 (annealed to because the trans-(R) and trans-(S) dA adducts intercalate from unmodified or adducted lower strands) or L7 (annealed to the major groove toward the 5⬘- and 3⬘-ends of the modified unmodified or adducted upper strands) were 5⬘-32P-labeled as strand, respectively (16 –20). Finally, we studied integrase inhibition by DKA inhibitors described above. After annealing, uracil DNA glycolylase was in the presence of BaP DE adducts to probe the drug-binding added to create an abasic site at the uracil position. The abasic site at the interface of the DNA and integrase. We discuss site leads to the formation of a Schiff base cross-link between these results in the following context: 1) inhibitor binding to the aldehyde group on the ribose and a nearby integrase lysine. the integrase active site; 2) insights into the interactions of The cross-links were stabilized by addition of 100 mM sodium integrase with the HIV U5 LTR; and 3) the DNA flexibility borohydride (final concentration). The cross-linked integrasethat is required for endonuclease cleavage (3⬘-P) to achieve a DNA products were separated from the substrate DNA by SDSproper transition state. PAGE using 16% Tricine gels (Invitrogen).

Integrase Accesses Supple DNA Backbones from the Minor Groove

RESULTS Effect of Sugar Modifications on Integrase Reactions—The sugar conformation of standard B-DNA exists mainly in C-2⬘endo conformation (south or s), whereas the less frequent A-form with a C-3⬘-endo conformation (north or n) is more typical of RNA (Fig. 1B). Oligonucleotides containing conformationally restricted sugar puckers at the site of integrase 3⬘-P were used to probe the conformational preferences of integrase during catalysis. Integrase recognizes the conserved 5⬘-CA in the HIV LTR (underlined in Fig. 1A). Adenosine analogs containing bicyclo[3.1.0]hexane (north or south, n or s), LNA, or ribonucleoside sugars were substituted for the conserved deoxyadenosine (Fig. 1C). The three conformationally constrained sugar modifications prevented integrase 3⬘-P, as indicated by the lack of 19-mer product (Fig. 1D, lanes 5, 6, and 10). The ribonucleoside permitted 3⬘-P and ST (Fig. 1D, lane 14), suggesting that the presence of the 2⬘-O functionality in LNA exhibits minimal interference with integrase catalysis. Note OCTOBER 27, 2006 • VOLUME 281 • NUMBER 43

that the cleaved ribo-containing oligonucleotides migrated slightly slower in the gel compared with fully deoxyribo-containing oligonucleotides, but a 20-mer band is present between the full-length and cleaved ribo-containing oligonucleotides, and therefore, we presume the altered migration is because of the extra hydroxyl group. These results show that conformational restrictions at the 3⬘-P site block 3⬘-P. Effect of Anomeric Inversion on Integrase 3⬘-P—The phosphodiester backbone conformation was further examined by placement of nucleotides containing ␣-anomerically inverted nucleotides at and around the site of integrase 3⬘-P. The normal B-DNA ␤-anomers connect via 5⬘–3⬘-phosphodiester linkages. The presence of a single ␣-anomer requires 3⬘–3⬘ and 5⬘–5⬘internucleotide linkages and a switch in the directionality of the DNA (Fig. 2, A and B) in order to permit Watson-Crick base pairing with an unmodified complementary strand. Substitution of the terminal GT with two ␣-anomers (Fig. 2, A and B, ␣1, 3⬘–3⬘ linkage) permitted a small amount of 3⬘-P (84% inhibiJOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 3. Structures of dA intercalating and dG minor groove BaP DE adducts. In both cases the absolute configuration at C-10 of the hydrocarbon (as shown) is S. A–C, dA intercalating adducts are attached to the adenine exocyclic N-6 amino group and intercalate into the DNA via the major groove on the 5⬘or 3⬘-sides of the adducted base for R or S stereoisomers, respectively (also see Fig. 4A). The structure is from Ref. 17; Protein Data Bank accession number is 1JDG. Note that this adduct derives from a different BaP DE diastereomer (benzylic hydroxyl group and epoxide oxygen cis) from the one used in this study. It was chosen for comparison to the U3(S) dA-adducted DNA because it is the only determined structure of a (10S)-BaP DE dA adduct paired with its normal T complement. The glycosidic conformation of the S dA adduct is syn. C, a stick representation of the dA adduct and the two surrounding bp is shown from above with the 5⬘-bp (including the adducted adenine on the right) in white and the 3⬘-bp in gray. The scissile phosphate is indicated by a star. D and E, dG adducts are attached to guanine 2-amino groups and occupy the minor groove, extending toward the 5⬘- or 3⬘-ends of the adducted strand for S (shown) or R stereoisomers, respectively (also see Fig. 4A). The structure is from Ref. 44; coordinates were provided by Dr. S. Broyde, New York University. In both structures, the adducted strand is shown in gray in the space-filled representations. Adducted bases are highlighted by white arrows. Phosphates are colored in red. The adducted base is not indicated in the minor groove view of the dG minor groove adduct because the base is behind the hydrocarbon adduct.

Integrase Accesses Supple DNA Backbones from the Minor Groove

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from the viral DNA end. For example, U3 refers to the 3rd base from the end of the upper strand and U3(S) is the isomer with 10S configuration located at the U3 position. Effect of Intercalating dA Adducts Attached to the Conserved Adenine on Integrase Reactions—The effect of site-specific DNA intercalators on integrase catalysis was probed using oligonucleotides containing a single BaP DE adduct attached to the conserved adenine (Fig. 3, A–C). NMR structures indicate that these adducts provide some bulk in the major groove and that the hydrocarbon stacks mainly with bases on the unadducted strand (see Fig. 3C from Ref. 17). Adducted DNAs were examined as full-length (Fig. 4A, upper sequences) and pre-cleaved (Fig. 4A, lower sequences, pc) duplex substrates. We showed previously that the lower strand L4(S) minor groove adduct blocks 3⬘-P and ST (Fig. 4B) (7). In contrast, intercalating BaP DE adducts attached to the conserved adenine (U3) had no affect on 3⬘-P and a partial inhibition of ST (55 FIGURE 4. Effect of dA intercalating and dG minor groove BaP DE adducts on HIV-1 integrase 3ⴕ-P and ST. and 60% inhibition, Fig. 4B). A similar A, sequences of full-length (upper duplexes) and precleaved (“pc”, lower duplexes) DNA substrates showing the location of dA intercalating (vertical bars) and dG minor groove (horizontal bars) adducts. B, representative gel effect on ST was observed for preshowing the effect of dA intercalating and dG minor groove BaP DE adducts on integrase reactions. Ctl indi- cleaved DNA. Complete inhibition of cates the unadducted DNA control. U3 BaP DE adducts retard electrophoretic migration. IN, integrase. ST by L4(S) adducted DNA, and partial inhibition of ST (58 and 67% inhition). The addition of a third ␣-anomer extending to the con- bition) by the U3 adducted DNAs was observed (Fig. 4B). These served adenine completely blocked 3⬘-P (Fig. 2, B and C, ␣2, results demonstrate that BaP DE adducts linked to the conserved 3⬘–3⬘ linkage). Finally, the presence of a single ␣-anomer sub- adenine reduce ST without affecting 3⬘-P. Importance of Functional Groups on the Conserved Adenine stitution for the conserved adenine also resulted in a complete block of 3⬘-P (Fig. 2, B and C, ␣3, 3⬘–3⬘ then 5⬘–5⬘ linkages). for Integrase Reactions—Because the dA adducts are attached Together with the results obtained using modified sugars, it is to the adenine exocyclic 6-amino group, the importance of the clear that the backbone structure around the 3⬘-P site is critical conserved adenine base was further evaluated through several base modifications (Fig. 5). 2-Aminopurine, lacking the 6-afor catalysis by integrase. Use of BaP DE Adducts as Molecular Probes—We next used mino group (Fig. 5B), was chosen to evaluate the importance of two types of BaP DE adducts to study the effects of DNA groove the point of attachment of the intercalating BaP DE adducts. occupancy and intercalation on integrase catalysis. Adducts 2-Aminopurine substitution resulted in no decrease in 3⬘-P and with 10R and 10S configuration at the point of attachment (Fig. only a slight decrease in ST (Fig. 5C, 14%). Hence, the exocyclic 3, C-10 of the hydrocarbon) to the exocyclic 6-amino group of N-6 group of the conserved adenine is not required for inteadenine intercalate on the 5⬘- or 3⬘- side of the adducted base, grase activity. Next, 7-deaza-adenine was chosen because of the respectively (Figs. 3, A–C, and 4A). The partially saturated ring possible importance of the adenine N-7 (9). Fig. 5C shows that linked to the adenine protrudes into the major groove and replacement of the adenine N-7 with carbon caused no change therefore provides major groove bulk that may potentially in integrase 3⬘-P and ST (Fig. 5C). In contrast, substitution of adenosine with an abasic (tetrahydrofuran) site resulted in a interact with integrase (Fig. 3, A–C). Adducts in which the hydrocarbon is attached to the exocy- partial (67%) inhibition of 3⬘-P and complete loss of ST (Fig. clic N-2 of guanine have the aromatic pyrene ring system 5C). It is important to note that this substitution probably located in the minor groove (Fig. 3, D and E) extending toward affects the backbone structure as well as base pairing. These the 5⬘- or 3⬘-end of the adducted strand for the S and R stereoi- results indicate that integrase tolerates modifications on the 2 somers, respectively (see Fig. 4A). By convention, the adducts and 6 positions of the conserved adenine but that removal of the will be referred to by their upper and lower strand positions base inhibits integrase activity.

Integrase Accesses Supple DNA Backbones from the Minor Groove

Effect of BaP DE Adducts Attached to the Terminal 5⬘-Adenine of the Lower Strand on Integrase Reactions—We next examined the effect of single BaP DE adducts attached to the 5⬘-terminal adenine of the unprocessed strand (Fig. 6A). These adducts may not be intercalated because of the terminal position of the base, especially following 3⬘-P. We found that the BaP DE adducts attached to the 5⬘-terminal adenine (L1) had no affect on 3⬘-P. However both the R and S BaP DE adducts inhibited ST (36 and 27% inhibition, respectively; see Fig. 6B). We further examined the pattern of ST by labeling each strand at the 3⬘-end with 32P to determine ST products specific for each strand of the acceptor duplex (7). Both the R and S adducts cause the formation of a strong 25-mer ST product corresponding to ST into the upper strand of the acceptor duplex DNA (Fig. 6C). ST into the lower strand of the acceptor DNA showed several new bands between 34- and 37-mer for the R stereoisomer (Fig. 6D). These results show that presence of a terminal BaP DE adduct does not affect 3⬘-P but alters the sequence specificity of ST. OCTOBER 27, 2006 • VOLUME 281 • NUMBER 43

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FIGURE 5. Effect of modifications for the conserved adenine on HIV-1 integrase 3ⴕ-P and ST. A, the location of adenine substitutions is underlined and the 3⬘-P site is indicated by a triangle. B, structures of adenine modifications examined. C, representative gel showing the affect of adenine substitutions on integrase reactions. IN, integrase.

Effect of Minor Groove BaP DE Adducts Linked to the 3⬘-Processed Dinucleotide on Integrase Reactions—We next examined the effect of single trans-opened BaP DE dG adducts attached to the guanine (U2) of the cleaved dinucleotide (Fig. 7A). This position was not addressed in our prior study (7). Therefore, we wished to extend our minor groove footprinting through the end of the DNA. The U2 minor groove adducts slightly inhibited 3⬘-P (Fig. 7, B and C). Because 3⬘-P presumably releases the terminal dinucleotide, we were surprised to observe a more pronounced inhibition of ST. To test whether the adducted dinucleotide could act as an ST inhibitor, we examined the effects of an adducted dinucleotide (5⬘-GT containing BaP DE adduct) added to integrase reactions performed with an unadducted (control) DNA substrate. Little inhibition of 3⬘-P or ST was observed (30% only at 333 ␮M; data not shown). These results demonstrate that BaP DE adducts on the U2 guanine reduce ST without affecting the efficiency of 3⬘-P, suggesting the retention of the adducted dinucleotide within the integrase-DNA complex following 3⬘-P (see “Discussion”). Use of BaP DE Adducts to Study Inhibition of Integrase by the Diketo Acid L-708906—Because diketo acid ST inhibitors have been hypothesized to bind at the enzyme-DNA interface at the end of the viral DNA (1, 23, 24) and because the BaP DNA adducts residing near the integrase 3⬘-P site decreased overall ST, which mimics the effect of DKAs, we examined the effect of these BaP DE adducts on inhibition of integrase by the DKA L-708906 (Fig. 8B). However, L-708906 is an ST-selective inhibitor of integrase with normal DNA (25) (Fig. 8, A–C). We observed a marked inhibition of 3⬘-P for the U2(S) dG-adducted DNA (Fig. 8, E and F, and Table 1), indicating this adduct increases the ability of L-708906 to act as a 3⬘-P inhibitor. Inhibition of the remaining ST was unchanged for the U2(S) dG-adducted DNA (Fig. 8E and Table 1), which is expected as 3⬘-P releases the adducted dinucleotide (Fig. 8D). In contrast, 3⬘-P inhibition by L-708906 was unaffected by a U3(S) dA adduct (Fig. 8H and Table 1). Furthermore, we observed an increase in the IC50 value for ST by L-708906 in the presence of the U3(S) dA adduct compared with unadducted DNA (Fig. 8H and Table 1), indicating that the U3(S) dA adduct interferes with ST inhibition by L-708906. By contrast, the terminal L1(S) dA adduct has no effect on integrase inhibition by L-708906 (Fig. 8K). Similar results were obtained with magnesium (data not shown). These results demonstrate that the presence of BaP DE adducts at specific positions affect integrase inhibition by L-708906. Effects of BaP DE Adducts on Inhibition of Integrase by MaDKA, L-870810, and L-Chicoric Acid—Because of the effects of the U3(S) dA and U2(S) dG adducts on inhibition of integrase by L-708906, we extended our studies to another ST-selective DKA inhibitor, Ma-DKA (4, 26). Similar results were observed and are summarized in Table 1. As for L-708906, the U2(S) dG adduct increased 3⬘-P inhibition by Ma-DKA (Fig. 8B, compare closed symbols), and the U3(S) dA adduct reduced ST inhibition by Ma-DKA (Fig. 8F, compare open symbols). The naphthyridine carboxamide L-870810 is also an ST-selective inhibitor like DKAs but induces different resistance mutations (27). The U2(S) dG adduct increased inhibition of

Integrase Accesses Supple DNA Backbones from the Minor Groove

FIGURE 7. Effect of dG minor groove BaP DE adducts within the cleaved dinucleotide on integrase catalysis. A, sequence of DNA substrate showing the location of the dG adducts. B, representative gel showing the effect of dG minor groove adducts on integrase 3⬘-P and ST. 21* indicates the retarded migration of the adducted oligonucleotide. Ctl indicates the unadducted control DNA. C, quantification of 3⬘-P and ST products for the U2 adducted DNA. The bars obtained for U2(R) are averaged from two experiments, and the bars obtained for U2(S) are from four separate experiments with error bars. Percent inhibition is relative to the unadducted control DNA. IN, integrase.

3⬘-P by L-870810, as was observed for the two DKAs (L-708906 and Ma-DKA). However, L-870810 was affected differently by the U3(S) dA BaP DE adduct compared with the two DKAs. The U3(S) dA adduct enhanced inhibition of 3⬘-P by L-870810 and

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DISCUSSION 3⬘-Processing by Integrase Requires DNA Backbone Flexibility —The flexibility of DNA probably plays an important role in enzymatic site-specific recognition and catalysis of DNA modifications by enzymes. For example, thymidine kinase preferentially phosphorylates its substrate thymidine in the south ribose conformation, whereas mammalian cellular DNA polymerase(s) shows a preference for the thymidine-5⬘-triphosphate in the north ribose conformation (30). North-restricted bicyclo[3.1.0]hexane-containing nucleoside triphosphates act as delayed chain terminators of reverse transcriptase when DNA synthesis approaches the A- to B-DNA transition point (31). LNA is RNase-resistant (32), and ␣-anomeric oligodeoxyribonucleotides are also resistant to endonuclease S1 and exonucleases (calf spleen phosphodiesterase and snake venom phosphodiesterase) (33). Obviously, conformational restriction of the phosphodiester backbone and anomeric inversion impart significant structural effects on nucleic acids. In our study, both north and south conformationally restricted sugars inhibited integrase 3⬘-P (Fig. 1). Note that manganese was used for these experiments, which is more “permissive” than magnesium for integrase reactions. Integrase was however able to bind to each duplex DNA containing a conformationally constrained substitution at a level similar to unmodified duplex, as measured by a Schiff base assay (22) (data not shown). Additionally, the ribose 2⬘-O-substitution of LNA is not inhibitory to integrase 3⬘-P as cleavage is observed in the presence of a ribonucleotide substitution. The ␣-anomer-containing duplex oligonucleotides were similarly resistant to integrase cleavage (Fig. 2), whereas binding integrase at a similar VOLUME 281 • NUMBER 43 • OCTOBER 27, 2006

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FIGURE 6. Effect of dA BaP DE adducts attached to the 5ⴕ-viral DNA end on integrase reactions. A, sequence of DNA substrate showing the location of the dA adducts. B, representative gel showing the effect of dA intercalating adducts on integrase 3⬘-P and ST. Ctl indicates the unadducted control DNA. C, representative gel showing the effect of L1 dA adducts on the pattern of upper strand ST products. The upper strand of the DNA was 3⬘-end-labeled with 32P, enabling the visualization of only upper strand ST products (7). The diagram below the gel shows the location of ST for the 25-mer ST product observed for both the R and S dA adducts. D, representative gel showing the effect of L1 adducts on the pattern of lower strand ST products. The lower strand of the DNA was 3⬘-end-labeled with 32P, enabling the visualization of only lower strand ST products. The diagram below the gel shows the location of ST for the 37-mer ST product observed for L1(R) dA-adducted DNA. IN, integrase.

reduced slightly the inhibition of ST, which is different from the DKAs (compare Fig. 9G with MaDKA in Fig. 9F and L-708906 in Fig. 8H). Hence, the naphthyridine carboxamide L-870810 exhibits differences for BaP DE adduct interference compared with the DKAs L-708906 and Ma-DKA. Finally, L-chicoric acid was chosen for comparison because of the following: 1) a G140S resistance mutation (28) does not overlap with DKA resistance mutations (25), and 2) because L-chicoric acid inhibits both 3⬘-P and ST (29), indicating a different binding site compared with DKAs. Similar levels of 3⬘-P and ST inhibition by L-chicoric acid were observed for both U2(S) dG- and U3(S) dA-adducted DNAs compared with unadducted DNA (Fig. 8, D and H). Together, these results demonstrate that BaP DE adducts can reveal site-specific differences in the inhibition sites for integrase inhibitors.

Integrase Accesses Supple DNA Backbones from the Minor Groove TABLE 1 IC50 valuesa for inhibition of integrase by inhibitors in the presence of BaP DE adducted oligonucleotides Inhibitor L-708906 Ma-DKA L-870810 L-Chicoric a

level as unmodified DNA was measured by Schiff base assay (data not shown). Nucleic acids containing pseudo sugars and anomeric inversion are known to be resistant to exo- and endonucleases (33, OCTOBER 27, 2006 • VOLUME 281 • NUMBER 43

3⬘-P ST 3⬘-P ST 3⬘-P ST 3⬘-P ST

DNA substrate 21/21

U2(S)/21

U3(S)/21

⬎12.3 0.04 ⬎12.3 0.23 ⬎12.3 0.05 0.19 0.30

0.4 0.04 5.2 0.17 6 0.05 0.39 0.30

5 0.50 ⬎12.3 6 ⬎12.3 0.03 0.12 0.29

Data were obtained from dose-response curves generated from three independent determinations (see Figs. 8 and 9). IC50 expressed in ␮M.

35, 36), but to our knowledge the underlying nuclease resistance mechanism has not been clarified. We propose that these backbone modifications may act by 1) altering the presentation and 2) restricting the flexibility of the scissile phosphate. A slight misalignment of the DNA within the active site of a nuclease could prevent the precise coordination of DNA and metal required for cleavage. Incorporation of an ␣-anomer into DNA necessitates addition or removal of one carbon atom because of the inverted directionality of the ␣ nucleotide. The resulting misalignment of the scissile phosphate may critically block nuclease cleavage. Restriction of phosphate flexibility propagated from a conformationally constrained sugar could inhibit inversion of the phosphate to achieve the bipyramidal transition state required for in-line attack by a nucleophile. For example, the 3⬘-hydroxyls of north and south bicyclo[3.1.0]hexane nucleotides are fixed equatorially and axially, respectively. In our case, that translates into a differential positioning of the leaving group connected to the scissile P–O bond without the flexibility of a normal deoxyribose. As the integrase reaction proceeds with inversion of configuration of the phosphate during 3⬘-P (37), the rigidity of the conformationally locked nucleosides may impose an energy barrier to achieving the pentacovalent intermediate where the nucleophile and leaving group are in the apical positions. In fact, this may be a general nuclease resistance mechanism for oligonucleotides such as LNA that are used in gene therapy and antisense techniques. Substitutions at the Conserved Adenine of the Viral DNA Still Allow 3⬘-P and ST—Our studies using oligonucleotides with constrained sugars and anomeric inversions demonstrate the importance of a flexible DNA backbone for integrase catalysis. Attachment of BaP adducts to the adenine of the conserved 5⬘-CA at position N-6 (Fig. 4) and removal of N-7 and exocyclic N-6 amino groups still allow 3⬘-P and ST (Fig. 5), indicating these groups are not required for integrase catalysis. Additionally, substitution of the conserved adenine with an abasic tetrahydrofuran resulted in a 67% inhibition of 3⬘-P (Fig. 5), both in the presence of manganese and magnesium (data not shown). Although we recognize that adenine must be important for integrase binding and catalysis, the tetrahydrofuran substitution probably also distorts the DNA backbone structure. Previous substitution of the conserved adenine with a propan-1,3diol residue (abasic hydrocarbon bridge (38 – 40)) resulting in a loss of 3⬘-P may in fact be reanalyzed in terms of backbone contacts with integrase. Hence, our results with substitution of JOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 8. Effect of BaP DE adducts on inhibition of integrase by the DKA L-708906. A, schematic diagram of integrase reactions with unadducted DNA. B, representative gel showing inhibition of integrase (IN) by L-708906 in the presence of unadducted DNA. C, schematic diagram of integrase reactions with DNA containing the U2(S) dG adduct. This diagram presumes that following 3⬘-P, the dinucleotide containing the BaP DE adduct is released, and the ST substrate does not contain an adduct. In graphs E, H, and K, 3⬘-P is indicated by filled symbols and ST by open symbols; unadducted DNA substrates are indicated by triangles and adducted substrates by squares. Note that these symbols are also indicated in the left panels. D, quantification of inhibition of integrase 3⬘P by L-708906 with and without a U2(S) dG adduct on the substrate DNA. E, schematic diagram of integrase reactions with DNA containing the U3(S) dA adduct. The ST substrate contains a BaP DE adduct. F, quantification of inhibition of integrase 3⬘-P by L-708906 with and without a U3(S) dA adduct on the substrate DNA. G, schematic diagram of integrase reactions with DNA containing the L1(S) dA adduct. The ST substrate contains a BaP DE adduct. H, quantification of inhibition of integrase 3⬘-P with and without a L1(S) dA adduct on the substrate DNA.

acid

Reaction

Integrase Accesses Supple DNA Backbones from the Minor Groove

the conserved adenine indicate that an intact adenine structure itself is not absolutely required for 3⬘-P and ST. Importance of Viral DNA Minor Groove Contacts at the 3⬘-P Site for Integrase Catalysis—Scanning of the viral LTR minor groove with BaP DE adducts illustrates specific contact regions for integrase activity. We previously examined the effects on integrase reactions of BaP DE adducts linked to the U5 and L4 guanines (7). We also examined the effect of each BaP adduct on integrase activity in the presence of magnesium and obtained similar results (data not shown). The U5(R), U5(S), and L4(S) adducts completely blocked 3⬘-P, and all stereoisomers at both positions blocked ST (7). We examined the U2 position in the present study to extend minor groove scanning in the vicinity of the 3⬘-P site. The U2 adduct still allowed efficient 3⬘-P and only partially reduced ST. Therefore, we suggest that integrase makes specific contacts with the viral LTR minor groove at the 5th and 4th bp from the DNA end, corresponding to 5⬘-GCAGT-3⬘. The minor groove closer to the tip of the DNA is not as important, as indicated by efficient 3⬘-P and ST observed with the U2-adducted oligonucleotides. In contrast to the block of integrase reactions by the U5 and L4 dG adducts, intercalating dA adducts linked to the conserved adenine (U3) and the terminal adenine (L1) permitted 3⬘-P (Figs. 4 and 6). Therefore, major groove interference and structural perturbations caused by intercalation appear to be well tolerated for 3⬘-P. Studies with human T-cell leukemia virus type 2 (HTLV-2) integrase suggest that this integrase binds specifically to the LTR DNA through minor groove contacts but requires major groove contacts for 3⬘-P and ST (41, 42). Therefore, the major groove may not be as important as the minor groove for viral DNA binding and 3⬘-P performed by HIV-1 compared with HTLV-2 integrase reactions. Further-

32436 JOURNAL OF BIOLOGICAL CHEMISTRY

more, NMR analysis of the terminal 17 bp of the HIV U5 LTR showed the following: 1) that the region near the cleavage site has a distorted minor groove and 2) base stacking between the L4 guanine and U3 adenine exposes the adenine 3⬘-hydroxyl in the minor groove for 3⬘-P, aiding in specific recognition by integrase (43). It is possible that the minor groove near the processing site interacts with integrase providing specificity of binding to the active site. The major groove would be exposed to solution during 3⬘-P, but may be more important during ST for contacts with other integrase subunits or the target DNA. Tolerance of BaP Adducts at the End of the Viral DNA Indicate That Integrase May Have an “Open” Active Site—The integrase active site may be open because we find that integrase can still function in the presence of U2 dG and U3 dA adducts. An open active site is reasonable because the target DNA needs space to approach the active site for the ST reaction. Release of the 5⬘-terminal dinucleotide may be required to create this open active site for ST. Although release of the dinucleotide is generally assumed to follow 3⬘-P, to our knowledge, this has not been shown experimentally. The ⬃60% loss of ST in the presence of the U2 dG adduct may indicate that the presence of the adduct on the dinucleotide prevents its release. The BaP DE adduct could bind to the enzyme active site following 3⬘-P and cause the dinucleotide to “stick” in the active site pocket (Fig. 10E), thereby prohibiting target DNA interaction and mimicking the ST inhibitors such as DKAs. Probing Integrase Inhibitor Binding in the Integrase Active Site—Separate integrase binding sites for donor (viral) and target DNA have been proposed, and inhibitors may bind differentially to these DNA-binding sites in order to inhibit selectively 3⬘-P and/or ST (1, 24). A 3⬘-P inhibitor (5CITEP, for example) may bind the donor DNA-binding site (24), whereas a VOLUME 281 • NUMBER 43 • OCTOBER 27, 2006

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FIGURE 9. Effect of BaP DE adducts on inhibition of integrase by Ma-DKA, L-870810, and L-chicoric acid. A, schematic diagram of integrase reactions with DNA containing the U2(S)-dG minor groove adduct. Presumably following 3⬘-P, the dinucleotide containing the BaP DE adduct is released. The ST substrate does not contain an adduct. B–D, quantification of inhibition of integrase 3⬘-P (filled symbols) and ST (open symbols) by Ma-DKA, L-870810, and L-chicoric acid, respectively, with unadducted (triangles) and U2(S) dG-adducted (squares) DNA substrates. E, schematic diagram of integrase reactions with DNA containing the U3(S) dA adduct. The ST substrate contains a BaP DE-dA adduct. F–H, quantification of inhibition of integrase 3⬘-P (filled symbols) and ST (open symbols) by Ma-DKA, L-870810 and L-chicoric acid, respectively, with unadducted (triangles) and U3(S) dA-adducted (squares) DNA substrates.

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binding to the ST complex but stabilizes interaction with L-870810. Together, our finding that BaP DE adducts modulate drug activities indicates that L-708906, Ma-DKA, and L-870810 interact with the integraseDNA complex at sites corresponding to the location of the BaP DE adducts. Furthermore, L-870810 binds integrase with different contacts compared with L-708906 and Ma-DKA, as was previously supported by unique resistance mutations compared with DKAs (27). By contrast, we observed no change in L-chicoric acid inhibition in the presence of the U2 dG- or U3 dA-adducted DNAs, indicating the L-chicoric acid-binding site does not overlap the binding sites for DKAs. This inhibitor induced a resistance mutation of integrase G140S in cell culture infected with HIV (28). Gly-140 is a hinge residue for the integrase flexible loop that resides over the active site (45). This resistance mutation does not arise after HIV exposure to DKA but confers resistance to the DKA L-731,988 (mechanistically similar to L-708906 (46)). These results suggest that interference assays with BaP DE-adducted DNA may be FIGURE 10. Proposed integrase interactions with DKA-like inhibitors in the presence of BaP DE adducts. used to investigate differential drugA and B, unadducted DNA: DKAs and naphthyridine carboxamides are selective inhibitors of ST. C–E, U2(S) binding sites for inhibitors with dG-adducted DNA: the adduct enhances inhibitor binding to the 3⬘-P complex. Approximately 40% of the DNA overlapping resistance profiles. undergoes ST. F and G, U3(S) dA-adducted DNA: the adduct blocks DKA binding (dashed line) but not L-870810 In summary, we found integrase binding (solid line) to the ST complex. Inhibition of 3⬘-P by L-870810 is enhanced by U3(S). The integrase active site and target DNA are indicated by a gray shaded box and a DNA helix, respectively. inhibition by conformationally restricted nucleosides and anomeric selective ST inhibitor (L-708906, for example) may bind the isomers placed near the 3⬘-P site, whereas integrase tolerates target DNA site at the interface of integrase, the two DNAs, and bulky BaP DE adducts in the same region. The apparent conthe two divalent metal ions (1). The U3(S) dA- and U2(S) dG- trast in the relatively small DNA backbone modifications that adducted DNA provided unique substrates to probe the bind- block catalysis with the large BaP DE adducts that permit cataling of inhibitors to the integrase active site. ysis suggests that integrase has specific backbone structure and We observed position-specific effects of the BaP DE adducts flexibility requirements in the viral DNA substrate. Inhibition on inhibition of integrase by three DKA-like compounds (Figs. of phosphate inversion during nuclease cleavage may be a gen8 and 9 and summarized in Fig. 10). The U2 dG adduct caused eral nuclease resistance mechanism by nucleic acids containing an increase in inhibitor effectiveness against 3⬘-P. Hence, this backbone modifications. Oligonucleotides containing site-speadduct probably modifies the 3⬘-P active site to provide a better cifically placed BaP DE adducts provide unique tools for exambinding site for DKAs that are normally ST-selective, resulting ination of inhibitor-binding sites at the integrase-DNA interin an increased inhibition of 3⬘-P (Fig. 10C). In contrast, the U3 face. BaP DE adduct interference has also proven useful with dA adduct selectively enhanced 3⬘-P inhibition for L-870810 other DNA-interacting enzymes, including DNA topoisomer(Fig. 10F), suggesting that L-870810 interacts differently with ases I (47–51) and II (16) and DNA polymerases (34, 52–55). the integrase 3⬘-P site compared with L-708906 and Ma-DKA. The U3 dA adduct also affected ST inhibition. This adduct decreased the effectiveness of L-708906 and Ma-DKA but Acknowledgments—We thank Dr. Kurt Kohn and Dr. Christophe increased the effectiveness of L-870810 against ST (Fig. 10G). Pre- Marchand for useful discussions and suggestions. sumably, the U3 dA adduct interferes with L-708906 and Ma-DKA

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