Effect of DNA Modifications on DNA Processing by HIV-1 Integrase and Inhibitor Binding
2006; Elsevier BV; Volume: 281; Issue: 43 Linguagem: Inglês
10.1074/jbc.m605101200
ISSN1083-351X
AutoresAllison A. Johnson, Jane M. Sayer, Haruhiko Yagi, Sachindra S. Patil, Françoise Debart, Martin A. Maier, David R. Corey, Jean‐Jacques Vasseur, Terrence R. Burke, Víctor E. Márquez, Donald M. Jerina, Yves Pommier,
Tópico(s)Cytomegalovirus and herpesvirus research
ResumoIntegration of the viral cDNA into host chromosomes is required for viral replication. Human immunodeficiency virus integrase catalyzes two sequential reactions, 3′-processing (3′-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 3′-processing site. 3′-Processing was blocked with substrates containing constrained sugars and α-anomeric residues, suggesting that integrase requires flexibility of the phosphodiester backbone at the 3′-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 3′-P site inhibited 3′-P, suggesting the importance of the minor groove contacts for 3′-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. Position-specific 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. Integration of the viral cDNA into host chromosomes is required for viral replication. Human immunodeficiency virus integrase catalyzes two sequential reactions, 3′-processing (3′-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 3′-processing site. 3′-Processing was blocked with substrates containing constrained sugars and α-anomeric residues, suggesting that integrase requires flexibility of the phosphodiester backbone at the 3′-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 3′-P site inhibited 3′-P, suggesting the importance of the minor groove contacts for 3′-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. Position-specific 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. HIV-1 2The 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. 2The 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. 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. 1Pommier Y. Johnson A.A. Marchand C. Nat. Rev. Drug Discov. 2005; 4: 236-248Crossref PubMed Scopus (638) Google Scholar, 2Van Maele B. Debyser Z. AIDS Rev. 2005; 7: 26-43PubMed Google Scholar, 3Lewinski M.K. Bushman F.D. Adv. Genet. 2005; 55: 147-181Crossref PubMed Scopus (121) Google Scholar, 4Li M. Mizuuchi M. Burke Jr., T.R. Craigie R. EMBO J. 2006; 25: 1295-1304Crossref PubMed Scopus (203) Google Scholar, 5Sinha S. Grandgenett D.P. J. Virol. 2005; 79: 8208-8216Crossref PubMed Scopus (71) Google Scholar.) 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 (6Esposito D. Craigie R. EMBO J. 1998; 17: 5832-5843Crossref PubMed Scopus (259) Google Scholar, 7Johnson A.A. Sayer J.M. Yagi H. Kalena G.P. Amin R. Jerina D.M. Pommier Y. J. Biol. Chem. 2004; 279: 7947-7955Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar, 8Mazumder A. Pommier Y. Nucleic Acids Res. 1995; 23: 2865-2871Crossref PubMed Scopus (26) Google Scholar). The conserved adenine, substituted by 5-iododeoxyuracil as a photocross-linker, forms a photocross-link to Lys-159 of integrase (9Jenkins T.M. Esposito D. Engelman A. Craigie R. EMBO J. 1997; 16: 6849-6859Crossref PubMed Scopus (214) Google Scholar). Residue Lys-159 also contacts the phosphate 5′ to the conserved deoxyadenosine (10Wang J.Y. Ling H. Yang W. Craigie R. EMBO J. 2001; 20: 7333-7343Crossref PubMed Scopus (311) Google Scholar). Mutagenesis showed that Tyr-143 and probably Gln-148 interact with the 5′-overhang resulting from 3′-P (9Jenkins T.M. Esposito D. Engelman A. Craigie R. EMBO J. 1997; 16: 6849-6859Crossref PubMed Scopus (214) Google Scholar, 11Ellison V. Brown P.O. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7316-7320Crossref PubMed Scopus (149) Google Scholar). 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) (12Gao K. Butler S.L. Bushman F. EMBO J. 2001; 20: 3565-3576Crossref PubMed Scopus (130) Google Scholar, 13Johnson A.A. Santos W. Pais G.C. Marchand C. Amin R. Burke Jr., T.R. Verdine G. Pommier Y. J. Biol. Chem. 2006; 281: 461-467Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Interactions between the backbone of the viral DNA and integrase have been sparsely examined. Phosphate ethylation interference was used to locate phosphates that are critical for integrase catalysis (14Bushman F.D. Craigie R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3458-3462Crossref PubMed Scopus (45) Google Scholar). Specific DNA backbone contacts required for ST near the insertion site were identified. On the cleaved strand of the viral DNA, the phosphate 5′ to the conserved adenine, as well as the two phosphates on the complementary strand that are closest to the cleavage site, were important for ST. The two phosphates at and following the ST site were important within the target DNA (14Bushman F.D. Craigie R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3458-3462Crossref PubMed Scopus (45) Google Scholar). 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 (7Johnson A.A. Sayer J.M. Yagi H. Kalena G.P. Amin R. Jerina D.M. Pommier Y. J. Biol. Chem. 2004; 279: 7947-7955Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar) and to create the most permissive conditions for integrase reactions. The data in Figs. 4, 5, 6, 7 were also performed with magnesium, with similar results (data not shown).FIGURE 5Effect of modifications for the conserved adenine on HIV-1 integrase 3′-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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6Effect of dA BaP DE adducts attached to the 5′-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 (7Johnson A.A. Sayer J.M. Yagi H. Kalena G.P. Amin R. Jerina D.M. Pommier Y. J. Biol. Chem. 2004; 279: 7947-7955Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 7Effect 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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 dinucleotide sequence to probe the DNA contacts between integrase and the viral DNA ends. We examined the effect of several DNA backbone modifications on integrase activity. Oligonucleotides containing conformationally constrained sugars attached to the conserved adenine were used to examine the conformational preference for north (north bicyclo[3.1.0]hexane and locked nucleic acid [LNA]) or south (south bicyclo[3.1.0]hexane) oriented sugars (Fig. 1C, see structures). A simple ribose substitution was also examined for comparison to LNA. Additionally, the effects of anomeric inversion on integrase 3′-P and ST were examined using oligonucleotides containing α-anomers around the 3′-P site (Fig. 2A). Substitution of an α-anomer results in a change in the normal 5′–3′ directionality of DNA. The presence of an α-anomer in DNA does not affect base pairing or duplex stability but can affect sugar puckering (15Aramini J.M. Kalisch B.W. Pon R.T. van de Sande J.H. Germann M.W. Biochemistry. 1996; 35: 9355-9365Crossref PubMed Scopus (31) Google Scholar). We also used covalent adducts derived from enantiomeric benzo[a]pyrene 7,8-diol 9,10-epoxides (BaP DE) attached to either single adenines or guanines to probe the position-specific effects of bulky adducts in the major or minor groove of the viral DNA on integrase 3′-P and ST. The hydrocarbon portion of the guanine N-2 adducts lies in the minor groove of the DNA and extends toward the 5′ or 3′ terminus of the adducted strand for the trans-(S) and trans-(R) adducts, respectively, where S and R refer to the absolute configuration at the point of attachment of the 2-amino group of the hydrocarbon (see Fig. 3, D and E) (7Johnson A.A. Sayer J.M. Yagi H. Kalena G.P. Amin R. Jerina D.M. Pommier Y. J. Biol. Chem. 2004; 279: 7947-7955Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar, 16Khan Q.A. Kohlhagen G. Marshall R. Austin C.A. Kalena G.P. Kroth H. Sayer J.M. Jerina D.M. Pommier Y. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12498-12503Crossref PubMed Scopus (40) Google Scholar) We reported previously that a BaP DE dG adduct in the minor groove of the 3′-P site blocked 3′-P (7Johnson A.A. Sayer J.M. Yagi H. Kalena G.P. Amin R. Jerina D.M. Pommier Y. J. Biol. Chem. 2004; 279: 7947-7955Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). Here we examined the effect of dG minor groove adducts attached to the 3′-processed dinucleotide, and we compared these results to those obtained with BaP DE adducts attached to the exocyclic N-6 amino group of adenines (Fig. 3, A–C). These adenine adducts were used to probe the DNA major groove and DNA unwinding on enzyme/DNA interactions because the trans-(R) and trans-(S) dA adducts intercalate from the major groove toward the 5′- and 3′-ends of the modified strand, respectively (16Khan Q.A. Kohlhagen G. Marshall R. Austin C.A. Kalena G.P. Kroth H. Sayer J.M. Jerina D.M. Pommier Y. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12498-12503Crossref PubMed Scopus (40) Google Scholar, 17Pradhan P. Tirumala S. Liu X. Sayer J.M. Jerina D.M. Yeh H.J. Biochemistry. 2001; 40: 5870-5881Crossref PubMed Scopus (46) Google Scholar, 18Volk D.E. Rice J.S. Luxon B.A. Yeh H.J. Liang C. Xie G. Sayer J.M. Jerina D.M. Gorenstein D.G. Biochemistry. 2000; 39: 14040-14053Crossref PubMed Scopus (52) Google Scholar, 19Yeh H.J. Sayer J.M. Liu X. Altieri A.S. Byrd R.A. Lakshman M.K. Yagi H. Schurter E.J. Gorenstein D.G. Jerina D.M. Biochemistry. 1995; 34: 13570-13581Crossref PubMed Scopus (80) Google Scholar, 20Zegar I.S. Chary P. Jabil R.J. Tamura P.J. Johansen T.N. Lloyd R.S. Harris C.M. Harris T.M. Stone M.P. Biochemistry. 1998; 37: 16516-16528Crossref PubMed Scopus (47) Google Scholar). Finally, we studied integrase inhibition by DKA inhibitors in the presence of BaP DE adducts to probe the drug-binding site at the interface of the DNA and integrase. We discuss these results in the following context: 1) inhibitor binding to the integrase active site; 2) insights into the interactions of integrase with the HIV U5 LTR; and 3) the DNA flexibility that is required for endonuclease cleavage (3′-P) to achieve a proper transition state. 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 ribose-containing 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, α-anomer, and BaP-modified oligonucleotides is described in the Supplemental Material. All oligonucleotides were further purified on denaturing 20% polyacrylamide gels. Single-stranded oligonucleotides were 5′-labeled using T4 polynucleotide kinase (Invitrogen) with [γ-32P]ATP (Amersham Biosciences) according to the manufacturers' instructions. Unincorporated nucleotide was removed by mini Quickspin oligo column (Roche Applied Science). The duplex DNA was annealed by addition of an equal concentration of the complementary strand, heating to 95 °C, and slow cooling to room temperature. Integrase Reactions—Recombinant wild-type HIV-1 integrase was purified from Escherichia coli as described (21Leh H. Brodin P. Bischerour J. Deprez E. Tauc P. Brochon J.C. LeCam E. Coulaud D. Auclair C. Mouscadet J.F. Biochemistry. 2000; 39: 9285-9294Crossref PubMed Scopus (120) Google Scholar) with the addition of 10% glycerol to all buffers. Integrase was incubated with DNA substrates for 1 h at 37°C. The reaction conditions were 500 nm integrase, 20 nm duplex DNA, 7.5 mm MnCl2, 5 mm NaCl, 14 mm 2-mercaptoethanol, and 20 mm MOPS, pH 7.2. Reactions were quenched by the addition of an equal volume of gel loading dye (formamide containing 1% SDS, 0.25% bromphenol blue and xylene cyanol). Products were separated on 20% polyacrylamide denaturing sequencing gels. Dried gels were visualized using a 445 SI PhosphorImager (Amersham Biosciences). Densitometric analysis was performed using ImageQuant software from Amersham Biosciences. Schiff Base Cross-linking Assay—The Schiff base cross-linking experiments were performed as described (22Mazumder A. Neamati N. Pilon A.A. Sunder S. Pommier Y. J. Biol. Chem. 1996; 271: 27330-27338Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Briefly, oligonucleotides containing uracil at position U3 (annealed to unmodified or adducted lower strands) or L7 (annealed to unmodified or adducted upper strands) were 5′-32P-labeled as described above. After annealing, uracil DNA glycolylase was added to create an abasic site at the uracil position. The abasic site leads to the formation of a Schiff base cross-link between the aldehyde group on the ribose and a nearby integrase lysine. The cross-links were stabilized by addition of 100 mm sodium borohydride (final concentration). The cross-linked integrase-DNA products were separated from the substrate DNA by SDS-PAGE using 16% Tricine gels (Invitrogen). 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 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 α-anomerically inverted nucleotides at and around the site of integrase 3′-P. The normal B-DNA β-anomers connect via 5′–3′-phosphodiester linkages. The presence of a single α-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 α-anomers (Fig. 2, A and B, α1, 3′–3′ linkage) permitted a small amount of 3′-P (84% inhibition). The addition of a third α-anomer extending to the conserved adenine completely blocked 3′-P (Fig. 2, B and C, α2, 3′–3′ linkage). Finally, the presence of a single α-anomer substitution for the conserved adenine also resulted in a complete block of 3′-P (Fig. 2, B and C, α3, 3′–3′ then 5′–5′ linkages). Together with the results obtained using modified sugars, it is clear that the backbone structure around the 3′-P site is critical for catalysis by integrase. Use of BaP DE Adducts as Molecular Probes—We next used two types of BaP DE adducts to study the effects of DNA groove occupancy and intercalation on integrase catalysis. Adducts with 10R and 10S configuration at the point of attachment (Fig. 3, C-10 of the hydrocarbon) to the exocyclic 6-amino group of adenine intercalate on the 5′- or 3′-side of the adducted base, respectively (Figs. 3, A–C, and 4A). The partially saturated ring linked to the adenine protrudes into the major groove and therefore provides major groove bulk that may potentially interact with integrase (Fig. 3, A–C). Adducts in which the hydrocarbon is attached to the exocyclic N-2 of guanine have the aromatic pyrene ring system located in the minor groove (Fig. 3, D and E) extending toward the 5′- or 3′-end of the adducted strand for the S and R stereoisomers, respectively (see Fig. 4A). By convention, the adducts will be referred to by their upper and lower strand positions 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. 17Pradhan P. Tirumala S. Liu X. Sayer J.M. Jerina D.M. Yeh H.J. Biochemistry. 2001; 40: 5870-5881Crossref PubMed Scopus (46) Google Scholar). 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) (7Johnson A.A. Sayer J.M. Yagi H. Kalena G.P. Amin R. Jerina D.M. Pommier Y. J. Biol. Chem. 2004; 279: 7947-7955Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). 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 and 60% inhibition, Fig. 4B). A similar effect on ST was observed for precleaved DNA. Complete inhibition of ST by L4(S) adducted DNA, and partial inhibition of ST (58 and 67% inhibition) by the U3 adducted DNAs was observed (Fig. 4B). These results demonstrate that BaP DE adducts linked to the conserved adenine reduce ST without affecting 3′-P. Importance of Functional Groups on the Conserved Adenine for Integrase Reactions—Because the dA adducts are attached to the adenine exocyclic 6-amino group, the importance of the conserved adenine base was further evaluated through several base modifications (Fig. 5). 2-Aminopurine, lacking the 6-amino group (Fig. 5B), was chosen to evaluate the importance of the point of attachment of the intercalating BaP DE adducts. 2-Aminopurine substitution resulted in no decrease in 3′-P and only a slight decrease in ST (Fig. 5C, 14%). Hence, the exocyclic N-6 group of the conserved adenine is not required for integrase activity. Next, 7-deaza-adenine was chosen because of the possible importance of the adenine N-7 (9Jenkins T.M. Esposito D. Engelman A. Craigie R. EMBO J. 1997; 16: 6849-6859Crossref PubMed Scopus (214) Google Scholar). Fig. 5C shows that replacement of the adenine N-7 with carbon caused no change in integrase 3′-P and ST (Fig. 5C). In contrast, substitution of adenosine with an abasic (tetrahydrofuran) site resulted in a partial (67%) inhibition of 3′-P and complete loss of ST (Fig. 5C). It is important to note that this substitution probably affects the backbone structure as well as base pairing. These results indicate that integrase tolerates modifications on the 2 and 6 positions of the conserved adenine but that removal of the base inhibits integrase activity. 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 (7Johnson A.A. Sayer J.M. Yagi H. Kalena G.P. Amin R. Jerina D.M. Pommier Y. J. Biol. Chem. 2004; 279: 7947-7955Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). 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. 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 (7Johnson A.A. Sayer J.M. Yagi H. Kalena G.P. Amin R. Jerina D.M. Pommier Y. J. Biol. Chem. 2004; 279: 7947-7955Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). 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 μm; 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 (1Pommier Y. Johnson A.A. Marchand C. Nat. Rev. Drug Discov. 2005; 4: 236-248Crossref PubMed Scopus (638) Google Scholar, 23Pommier Y. Marchand C. Curr. Med. Chem. Anticancer Agents. 2005; 5: 421-429Crossref PubMed Scopus (64) Google Scholar, 24Marchand C. Zhang X. Pais G.C. Cowansage K. Neamati N. Burke Jr., T.R. Pommier Y. J. Biol. Chem. 2002; 277: 12596-12603Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) 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 (25Hazuda D.J. Felock P. Witmer M. Wolfe A. Stillmock K. Grobler J.A. Espesath A. Gabryelski L. Schlelf W. Blau C. Miller M.D. Science. 2000; 287: 646-650Crossref PubMed Scopus (1065) Google Scholar) (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
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