Noncleaved Strand Research Articles

Discovery of Novel Integrase Inhibitors Acting outside the Active Site Through High-Throughput Screening.

Currently, an increasing number of drugs are becoming available to clinics for the treatment of HIV infection. Even if this targeted therapy is highly effective at suppressing viral replication, caregivers are facing growing therapeutic failures in patients, due to resistance with or without treatment adherence concerns. Accordingly, it is important to continue to discover small molecules that have a novel mechanism of inhibition. In this work, HIV integrase inhibitors were selected by high-throughput screening. Chemical structure comparisons enabled the identification of stilbene disulfonic acids as a potential new chemotype. Biochemical characterization of the lead compound stilbenavir (NSC34931) and a few derivatives was performed. Stilbene disulfonic acid derivatives exhibit low to sub-micromolar antiviral activity, and they inhibit integrase through DNA-binding inhibition. They probably bind to the C-terminal domain of integrase, in the cavity normally occupied by the noncleaved strand of the viral DNA substrate. Because of this original mode of action compared to active site strand transfer inhibitors, they do not exhibit cross-resistance to the three main resistance pathways to integrase inhibitors (G140S-Q148H, N155H, and Y143R). Further structure–activity optimization should enable the development of more active and less toxic derivatives with potential clinical relevance.

Architecture and Assembly of HIV Integrase Multimers in the Absence of DNA Substrates

We have applied small angle x-ray scattering and protein cross-linking coupled with mass spectrometry to determine the architectures of full-length HIV integrase (IN) dimers in solution. By blocking interactions that stabilize either a core-core domain interface or N-terminal domain intermolecular contacts, we show that full-length HIV IN can form two dimer types. One is an expected dimer, characterized by interactions between two catalytic core domains. The other dimer is stabilized by interactions of the N-terminal domain of one monomer with the C-terminal domain and catalytic core domain of the second monomer as well as direct interactions between the two C-terminal domains. This organization is similar to the "reaching dimer" previously described for wild type ASV apoIN and resembles the inner, substrate binding dimer in the crystal structure of the PFV intasome. Results from our small angle x-ray scattering and modeling studies indicate that in the absence of its DNA substrate, the HIV IN tetramer assembles as two stacked reaching dimers that are stabilized by core-core interactions. These models of full-length HIV IN provide new insight into multimer assembly and suggest additional approaches for enzyme inhibition.

Retroviral Integrases Promote Fraying of Viral DNA Ends

In the initial step of integration, retroviral integrase (IN) introduces precise nicks in the degenerate, short inverted repeats at the ends of linear viral DNA. The scissile phosphodiester bond is located immediately 3' of a highly conserved CA/GT dinucleotide, usually 2 bp from the ends. These nicks create new recessed 3'-OH viral DNA ends that are required for joining to host cell DNA. Previous studies have indicated that unpairing, "fraying," of the viral DNA ends by IN contributes to end recognition or catalysis. Here, we report that end fraying can be detected independently of catalysis with both avian sarcoma virus (ASV) and human immunodeficiency virus type 1 (HIV-1) IN proteins by use of fluorescence resonance energy transfer (FRET). The results were indicative of an IN-induced intramolecular conformational change in the viral DNA ends (cis FRET). Fraying activity is tightly coupled to the DNA binding capabilities of these enzymes, as follows: an inhibitor effective against both IN proteins was shown to block ASV IN DNA binding and end fraying, with similar dose responses; ASV IN substitutions that reduced DNA binding also reduced end fraying activity; and HIV-1 IN DNA binding and end fraying were both undetectable in the absence of a metal cofactor. Consistent with our previous results, end fraying is sequence-independent, suggesting that the DNA terminus per se is a major structural determinant for recognition. We conclude that frayed ends represent a functional intermediate in which DNA termini can be sampled for suitability for endonucleolytic processing.

Insights from the structure of a smallpox virus topoisomerase-DNA transition state mimic.

Poxviruses encode their own type IB topoisomerases (TopIBs), which release superhelical tension generated by replication and transcription of their genomes. To investigate the reaction catalyzed by viral TopIBs, we have determined the structure of a variola virus topoisomerase-DNA complex trapped as a vanadate transition state mimic. The structure reveals how the viral TopIB enzymes are likely to position the DNA duplex for ligation following relaxation of supercoils and identifies the sources of friction observed in single-molecule experiments that argue against free rotation. The structure also identifies a conformational change in the leaving group sugar that must occur prior to cleavage and reveals a mechanism for promoting ligation following relaxation of supercoils that involves an Asp-minor groove interaction. Overall, the new structural data support a common catalytic mechanism for the TopIB superfamily but indicate distinct methods for controlling duplex rotation in the small versus large enzyme subfamilies.

Oligonucleotide cleavage and rejoining by topoisomerase III from the hyperthermophilic archaeon Sulfolobus solfataricus: temperature dependence and strand annealing‐promoted DNA religation

Topoisomerase III from the hyperthermophilic archaeon Sulfolobus solfataricus (Sso topo III) is optimally active in DNA relaxation at 75 degrees C. We report here that Sso topo III-catalysed DNA cleavage and religation differed significantly in temperature dependence: the enzyme was most active in cleaving ssDNA containing a cleavage site at 25-50 degrees C, but was efficient in rejoining the cleaved DNA strand only at higher temperatures (e.g. > or = 45 degrees C). The failure of Sso topo III to rejoin the cleaved DNA strand efficiently appeared to be responsible for the inability of the enzyme to relax negatively supercoiled DNA at low temperature (e.g. 25 degrees C). Intriguingly, Sso topo III facilitated DNA annealing although it showed higher affinity for ssDNA than for dsDNA. Religation of the DNA strand cleaved by Sso topo III was drastically enhanced when the DNA was allowed to anneal to a complementary non-cleaved oligonucleotide, presumably as a result of destabilization of the interaction between the enzyme and the cleaved strand through the formation of duplex DNA. A region in the non-cleaved strand corresponding to a sequence containing six bases on the 5' side and two bases on the 3' side of the cleavage site in the cleaved strand was crucial to the annealing-promoted religation. However, the annealing-promoted religation was relatively insensitive to mismatches in this region and the region conserved for oligonucleotide cleavage, except for that at the 5' end of the broken strand. These results suggest that Sso topo III is well suited for a role in DNA rewinding, whether it leads to homoduplex or heteroduplex formation.

Site-specific DNA transesterification by vaccinia topoisomerase: role of specific phosphates and nucleosides.

Vaccinia topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a pentapyrimidine target site 5'-CCCTTp downward arrow in duplex DNA. Here we present experiments that illuminate the contributions of specific nucleosides and phosphates to site affinity and transesterification. We find that the -1 phosphate and -2 nucleoside on the scissile strand (5'-CCCTTp / NpN) enhance the rate of transesterification by factors of 40 and 25, respectively, whereas the DNA segment downstream of the -2 nucleotide makes no significant kinetic contribution. Placement of a 5'-phosphate/3'-OH nick at position +2, +3, +4, or +5 within the CCCTT element results in a 5-10-fold reduction in the affinity of topoisomerase binding to DNA. A nick at the +2 phosphate also slows the rate of transesterification by approximately 500-fold. This finding, together with earlier studies of the effects of position-specific base and sugar modifications, points to the +2 Tp nucleotide as being the most critical element of the CCCTT target site other than the scissile phosphate itself. On the noncleaved strand, the segment downstream of the 3'-GGGAA element contributes minimally to the rate of transesterification provided that the substrate is otherwise fully base-paired within the 5'-CCCTT target site. By studying the effects of single nucleotide gaps and missing phosphate nicks within the 3'-GGGAA sequence, we find that the +1 and +2 adenosine nucleosides enhance the rate of transesterification by 20- and 1,000-fold respectively and that the +5 phosphate (3'-GpGGAA) is also important for cleavage. Cumulative functional analyses of the vaccinia topoisomerase-DNA interface are discussed in light of newly available structures for the vaccinia and human type IB enzymes.

DNA Duplexes Containing 3′-Deoxynucleotides as Substrates for DNA Topoisomerase I Cleavage and Ligation

The DNA cleavage-ligation reaction of DNA topoisomerase I was investigated employing synthetic DNA substrates containing 3'-deoxyadenosine or 3'-deoxythymidine at specific sites and acceptor oligonucleotides of different lengths. The modified nucleotides were substituted systematically within the putative enzyme-binding domain and also next to the high efficiency cleavage site to determine the effect of single base changes on enzyme function. Depending on the site of substitution, the facility of the cleavage and ligation reactions were altered. The bases at positions -1 and -2 on the noncleaved strand were found to be important for determining the site of cleavage. Inclusion of 3'-deoxythymidine in the scissile strand at position -1 permitted the demonstration that topoisomerase I can cleave and form a 2' --> 5'-phosphodiester linkage. Partial duplexes doubly modified at positions -4 or -6 in the noncleaved strand and at positions +1 or -1 within scissile strand were not good substrates for topoisomerase I, showing that cleavage can depend importantly on binding interactions based on structural alterations at spatially separated sites. Substitution of a 3'-deoxynucleotide on the scissile strand at position -6 enhanced formation of the ligation product resulting from cleavage at site 1 and suppressed cleavage at site 2.

Probing interactions between viral DNA and human immunodeficiency virus type 1 integrase using dinucleotides.

Retroviral integrases are essential for viral replication and represent an attractive chemotherapeutic target. In the current study, we demonstrated the activity of micromolar concentrations of dinucleotides against human immunodeficiency virus type 1 (HIV-1), HIV type 2 (HIV-2), simian immunodeficiency virus, and feline immunodeficiency virus integrases. The structure-activity relationship indicates that 5'-phosphorylation enhances potency and that phosphodiester and sugar modifications affect the inhibition of HIV-1 integrase. Base sequence selectivity was observed: pAC, pAT, and pCT were the most potent inhibitors, whereas pAA, pGA, and pGC showed low activity at 100 microM. The inhibition by pAC is consistent with the interaction of the enzyme with the 5' end of the noncleaved strand (5'-AC-3'). The linear and cyclic dinucleotides released by the 3'-processing reaction did not affect enzymatic activity at physiological concentrations. An increase in the length to trinucleotides or tetranucleotides enhanced potency by only 2-3-fold, suggesting that two neighboring bases may be sufficient for significant interactions. Inhibition of a truncated (50-212) integrase mutant and global inhibition of all nucleophiles in the 3'-processing reaction suggest that dinucleotides bind in the catalytic core. All of the active dinucleotides inhibited enzyme/DNA binding in their respective IC50 range. Although the dinucleotides tested showed no antiviral activity, these observations demonstrate the usefulness of dinucleotides in elucidating enzyme mechanisms and as potential ligands for cocrystallization and as lead structures for development of antivirals.

The effect of camptothecin on topoisomerase I catalysis.

The existence of a covalent intermediate in topoisomerase I catalysis allows uncoupling of the cleavage and ligation half-reactions on partially single-stranded DNA substrates containing a highly preferred interaction site. Using this model DNA substrate system we have demonstrated that the cleavage reaction requires bipartite interaction with two distinct DNA duplex regions; One located around the cleavage site (region A) and another located on the side holding the 5'-OH end generated by cleavage (region B). The postcleavage complexes containing the enzyme covalently attached at an internal position are capable of ligating DNA strands matching the noncleaved strand. Previously, we have characterized the effect of the antitumor agent camptothecin on the two half-reactions of topoisomerase I catalysis on DNA substrates allowing bipartite DNA interaction. The obtained results demonstrated that the drug only inhibited the ligation reaction leaving the cleavage reaction unaffected at the studied site. Here, we report that camptothecin also impairs ligation of the cleaved strand to a dinucleotide within region A in the absence of additional DNA contacts. When these results are taken together with the observation that camptothecin-trapped topoisomerase I-DNA complexes preferentially are generated at sites containing guanine immediately 3' to the cleavage position, it suggests that camptothecin inhibits the ligation reaction by forming a reversible ternary complex with the enzyme and DNA at the cleavage site within region A.

Alteration of DNA Primary Structure by DNA Topoisomerase I. Isolation of the Covalent Topoisomerase I−DNA Binary Complex in Enzymatically Competent Form

DNA ligation by DNA topoisomerase I was investigated employing synthetic DNA substrates containing a single strand nick. Site-specific cleavage of the DNA by topoisomerase I in proximity to the nick resulted in uncoupling of the cleavage and ligation reactions of the enzyme, thereby trapping the covalent enzyme−DNA intermediate. DNA cleavage could be reversed by the addition of acceptor oligonucleotides containing a free 5‘-OH group and capable of hybridizing to the noncleaved strand of the “suicide substrates”. Utilizing acceptors with partial complementarity, modification of nucleic acid structure has been obtained. Modifications included the formation of DNA insertions, deletions, and mismatches. To further evaluate the potential of topoisomerase I to mediate structural transformations of DNA, acceptor oligonucleotides containing nucleophiles other than OH groups at the 5‘-end were studied as substrates for the topoisomerase I-mediated ligation reaction. Toward this end, oligonucleotides containing 5‘-...

Vaccinia topoisomerase binds circumferentially to DNA.

Vaccinia DNA topoisomerase, a member of the eukaryotic type I enzyme family, binds duplex DNA and forms a covalent adduct at sites containing a conserved sequence element 5'-CCCTT decreases in the scissile strand. The protein-DNA interface entails essential contacts with four phosphate moieties within the CCCTT motif, including the scissile phosphate, and three phosphates within the GGGAA sequence on the noncleaved strand. Critical protein-phosphate contacts are arrayed across the minor groove of the DNA helix. Base-specific contacts with the pentamer element are within the major groove and are situated on the opposite face of the helix. Thus, vaccinia topoisomerase binds circumferentially to its target site in duplex DNA. This binding mode suggests that the eukaryotic enzyme adopts a toroidal shape in the DNA-bound state. Conformational isomerization of the bound protein provides a plausible mechanism for DNA relaxation.

Eukaryotic topoisomerase I-mediated cleavage requires bipartite DNA interaction. Cleavage of DNA substrates containing strand interruptions implicates a role for topoisomerase I in illegitimate recombination

Topoisomerase I-mediated cleavage has previously been demonstrated to require interaction of the enzyme with a DNA duplex region encompassing the cleavage site (Svejstrup, J. Q., Christiansen, K., Andersen, A. H., Lund, R., and Westergaard, O (1990) J. Biol. Chem. 265, 12529-12535). The required region, designated region A, includes positions -5 through -1 on the noncleaved strand and positions -7 through +2 on the scissile strand, relative to the cleavage site. Utilizing defined DNA substrates in topoisomerase I cleavage assays we show that efficient cleavage within region A requires additional interaction of the enzyme with duplex DNA on the side holding the 5'-OH end generated by cleavage. By analyzing the interaction of topoisomerase I with DNA substrates varying by single nucleotides on either strand outside region A, an additional duplex region, designated region B, was delimited to positions 6-11. The ability of topoisomerase I to interact separately with regions A and B was assayed on sets of DNA substrates containing a nested series of single-stranded branch sites. The obtained results demonstrate that the normal reversible cleavage/religation equilibrium established by topoisomerase I on continuous duplex DNA is replaced by irreversible cleavage on DNA substrates containing branch sites between the cleavage site and region B as these DNA substrates allow cleavage but prevent religation due to release of the incised strands. The intramolecular bipartite interaction mode of topoisomerase I during the cleavage reaction is thus indicated by both the absence of enzyme-mediated duplex stabilization and the wide tolerance for protruding strands between the cleavage site and region B. Since the irreversibly cleaved topoisomerase I-DNA complexes are kinetically competent to ligate added DNA fragments carrying free 5'-OH ends, the results suggest a role of topoisomerase I in illegitimate recombination.

DNA strand transfer reactions catalyzed by vaccinia topoisomerase I.

Vaccinia virus DNA topoisomerase I forms a 3'-phosphoryl intermediate with duplex DNAs containing the conserved binding/cleavage motif 5'CCCTT decreases. Covalently bound enzyme is capable of transferring the incised DNA strand to a heterologous DNA acceptor containing a 5'OH terminus. Both intramolecular and intermolecular religation reactions are catalyzed. Intramolecular strand transfer occurs to the noncleaved strand of the DNA duplex and results in formation of a hairpin loop. Intermolecular religation to an exogenous DNA strand is favored over hairpin formation and requires the potential for base pairing between the acceptor and the noncleaved strand of the donor complex. As few as 4 potential base pairs are sufficient to support intermolecular transfer. These results in vitro are consistent with the proposal that vaccinia topoisomerase can catalyze sequence-specific strand transfer during genetic recombination in vivo (Shuman, S. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 10104-10108.).

New technique for uncoupling the cleavage and religation reactions of Eukaryotic Topoisomerase I.: The mode of action of camptothecin at a specific recognition site

A new technique for uncoupling the cleavage and religation half-reactions of topoisomerase I at a specific site has been developed. The technique takes advantage of a suicidal DNA substrate to attain enzyme-mediated cleavage without concomitant religation. Efficient religation can be achieved, subsequently, by addition of an oligonucleotide capable of hybridising to the non-cleaved strand of the suicide DNA substrate. The technique was used to study the effect of different compounds on the half-reactions of topoisomerase I. It was shown that topoisomerase I-mediated cleavage was inhibited by NaCl concentrations higher than 200 m m, while the religation reaction seemed unaffected by concentrations as high as 3 m-NaCl. The divalent cations Mg 2+, Ca 2+ and Mn 2+ were found to enhance the cleavage but not the religation reaction of topoisomerase I, whereas Cu 2+ and Zn 2+ inhibited both reactions. Furthermore, the effect of the antineoplastic agent, camptothecin, on the half-reactions of topoisomerase I was investigated. It was found that the drug did not affect the cleavage reaction of topoisomerase I at the studied site, while the religation reaction of the enzyme was inhibited. Camptothecin was found to stabilise the enzyme-DNA cleavage complex even when the drug was added after complex formation.

Site-specific interaction of vaccinia virus topoisomerase I with duplex DNA. Minimal DNA substrate for strand cleavage in vitro.

Purified vaccinia virus DNA topoisomerase I forms a cleavable complex with duplex DNA at a conserved sequence element 5'(C/T)CCTTdecreases in the incised DNA strand. DNase I footprint studies show that vaccinia topoisomerase protects the region around the site of covalent adduct formation from nuclease digestion. On the cleaved DNA strand, the protected region extends from +13 to -13 (+1 being the site of cleavage). On the noncleaved strand, the protected region extends from +13 to -9. Similar nuclease protection is observed for a mutant topoisomerase (containing a Tyr ---- Phe substitution at the active site amino acid 274) that is catalytically inert and does not form the covalent intermediate. Thus, vaccinia topoisomerase is a specific DNA binding protein independent of its competence in transesterification. By studying the cleavage of a series of 12-mer DNA duplexes in which the position of the CCCTTdecreases motif within the substrate is systematically phased, the "minimal" substrate for cleavage has been defined; cleavage requires six nucleotides upstream of the cleavage site and two nucleotides downstream of the site. An analysis of the cleavage of oligomer substrates mutated singly in the CCCTT sequence reveals a hierarchy of mutational effects based on position within the pentamer motif and the nature of the sequence alteration.

Site-specific interaction of vaccinia virus topoisomerase I with duplex DNA. Minimal DNA substrate for strand cleavage in vitro

Purified vaccinia virus DNA topoisomerase I forms a cleavable complex with duplex DNA at a conserved sequence element 5'(C/T)CCTTdecreases in the incised DNA strand. DNase I footprint studies show that vaccinia topoisomerase protects the region around the site of covalent adduct formation from nuclease digestion. On the cleaved DNA strand, the protected region extends from +13 to -13 (+1 being the site of cleavage). On the noncleaved strand, the protected region extends from +13 to -9. Similar nuclease protection is observed for a mutant topoisomerase (containing a Tyr ---- Phe substitution at the active site amino acid 274) that is catalytically inert and does not form the covalent intermediate. Thus, vaccinia topoisomerase is a specific DNA binding protein independent of its competence in transesterification. By studying the cleavage of a series of 12-mer DNA duplexes in which the position of the CCCTTdecreases motif within the substrate is systematically phased, the minimal substrate for cleavage has been defined; cleavage requires six nucleotides upstream of the cleavage site and two nucleotides downstream of the site. An analysis of the cleavage of oligomer substrates mutated singly in the CCCTT sequence reveals a hierarchy of mutational effects based on position within the pentamer motif and the nature of the sequence alteration.

Site-specific DNA cleavage by vaccinia virus DNA topoisomerase I. Role of nucleotide sequence and DNA secondary structure

Cleavage of linear duplex DNA by purified vaccinia virus DNA topoisomerase I occurs at a conserved sequence element (5'-C/T)CCTT decreases) in the incised DNA strand. Oligonucleotides spanning the high affinity cleavage site CCCTT at nucleotide 2457 in pUC19 DNA are cleaved efficiently in vitro, but only when hybridized to a complementary DNA molecule. As few as 6 nucleotides proximal to the cleavage site and 6 nucleotides downstream of the site are sufficient to support exclusive cleavage at the high affinity site (position +1). Single nucleotide substitutions within the consensus pentamer have deleterious effects on the equilibria of the topoisomerase binding and DNA cleavage reactions. The effects of base mismatch within the pentamer are more dramatic than are the effects of mutations that preserve base complementarity. Competition experiments indicate that topoisomerase binds preferentially to DNA sites containing the wild-type pentamer element. Single-stranded DNA containing the sequence CCCTT in the cleaved stand is a more effective competitor than is single-stranded DNA containing the complementary sequence in the noncleaved strand.

Minimal DNA duplex requirements for topoisomerase I-mediated cleavage in vitro.

The minimal DNA duplex requirements for topoisomerase I-mediated cleavage at a specific binding sequence were determined by analyzing the interaction of the enzyme with sets of DNA substrates varying successively by single nucleotides at the 5'- or 3' end of either strand. Topoisomerase I cleavage experiments showed a minimal region of nine nucleotides on the scissile strand and five nucleotides on the noncleaved strand. On the scissile strand, seven of the nine nucleotides were situated upstream to the cleavage site, while all five nucleotides required on the non-cleaved strand were located to this side. The results suggested that topoisomerase I bound tightly to this region, stabilizing the DNA duplex extensively. On minimal substrates which were partially single-stranded downstream to the cleavage site, cleavage was suicidal, that is, the enzyme was able to cleave the substrates, but unable to perform the final religation.