Biomedical subjects

Marina Gottikh

Publications and source records attributed to Marina Gottikh.

7 recordsLinked to original sources

PubMed2006

Relationship between the oligomeric status of HIV-1 integrase on DNA and enzymatic activity.

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

Anisotropy↗

PubMed2006

Probing of HIV-1 integrase/DNA interactions using novel analogs of viral DNA.

The specific activity of the human immunodeficiency virus, type 1 (HIV-1), integrase on the viral long terminal repeat requires the binding of the enzyme to certain sequences located in the U3 and U5 regions at the ends of viral DNA, but the determinants of this specific DNA-protein recognition are not yet completely understood. We synthesized DNA duplexes mimicking the U5 region and containing either 2'-modified nucleosides or 1,3-propanediol insertions and studied their interactions with HIV-1 integrase, using Mn2+ or Mg2+ ions as integrase cofactors. These DNA modifications had no strong effect on integrase binding to the substrate analogs but significantly affected 3'-end processing rate. The effects of nucleoside modifications at positions 5, 6, and especially 3 strongly depended on the cationic cofactor used. These effects were much more pronounced in the presence of Mg2+ than in the presence of Mn2+. Modifications of base pairs 7-9 affected 3'-end processing equally in the presence of both ions. Adenine from the 3rd bp is thought to form at least two hydrogen bonds with integrase that are crucial for specific DNA recognition. The complementary base, thymine, is not important for integrase activity. For other positions, our results suggest that integrase recognizes a fine structure of the sugar-phosphate backbone rather than heterocyclic bases. Integrase interactions with the unprocessed strand at positions 5-8 are more important than interactions with the processed strand for specific substrate recognition. Based on our results, we suggest a model for integrase interaction with the U5 substrate.

Base Pairing↗

PubMed2006

Kinetic study of the HIV-1 DNA 3'-end processing.

The 3'-processing of viral DNA extremities is the first step in the integration process catalysed by human immunodeficiency virus (HIV)-1 integrase (IN). This reaction is relatively inefficient and processed DNAs are usually detected in vitro under conditions of excess enzyme. Despite such experimental conditions, steady-state Michaelis-Menten formalism is often applied to calculate characteristic equilibrium/kinetic constants of IN. We found that the amount of processed product was not significantly affected under conditions of excess DNA substrate, indicating that IN has a limited turnover for DNA cleavage. Therefore, IN works principally in a single-turnover mode and is intrinsically very slow (single-turnover rate constant = 0.004 min(-1)), suggesting that IN activity is mainly limited at the chemistry step or at a stage that precedes chemistry. Moreover, fluorescence experiments showed that IN-DNA product complexes were very stable over the time-course of the reaction. Binding isotherms of IN to DNA substrate and product also indicate tight binding of IN to the reaction product. Therefore, the slow cleavage rate and limited product release prevent or greatly reduce subsequent turnover. Nevertheless, the time-course of product formation approximates to a straight line for 90 min (apparent initial velocity), but we show that this linear phase is due to the slow single-turnover rate constant and does not indicate steady-state multiple turnover. Finally, our data ruled out the possibility that there were large amounts of inactive proteins or dead-end complexes in the assay. Most of complexes initially formed were active although dramatically slow.

DNA, Viral↗

PubMed2005

In vivo potentialities of EWS-Fli-1 targeted antisense oligonucleotides-nanospheres complexes.

The EWS/FLI-1 fusion gene, resulting from a t(11;22) translocation, plays a key role in the pathogenesis of Ewing sarcoma. Previously, we have shown that antisense oligonucleotides designed against EWS-Fli-1 inhibited tumor growth in nude mice provided they were delivered intratumorally by nanocapsules or by CTAB-coated nanospheres. In this study, we have used two types of nanospheres (designated as type 1 and type 2 nanospheres) stabilized with chitosan for both intratumoral and systemic administration of oligonucleotides. Inhibition of the tumor growth in vivo was found to be dependent on the carrier type as well as on antisense oligonucleotide modification. Indeed, whereas both types of nanospheres were efficient in reducing tumor growth after intratumoral injection, we have obtained only with type 2 nanospheres an antitumoral effect after intravenous injection in a preliminary experiment. Additionally, the anticancer efficacy of a localized modification of the EWS-Fli-1 phosphodiester/phosphorothioate chimeric antisense oligonucleotide was demonstrated. In cell culture the oligonucleotides inhibit cell growth by their antisense activity. Further investigations are needed in vivo to learn the mechanism of action of the complexes.

Animals↗

PubMed2004

HIV-1 integrase complexes with DNA dissociate in the presence of short oligonucleotides conjugated to acridine.

The human immunodeficiency virus type 1 (HIV-1) integrase is an essential enzyme in the life cycle of the virus and is therefore an attractive target for the development of new antiviral drugs. Among them, inhibitors which are capable of targeting the preassembled integrase/DNA complex are of particular interest, because they could suppress integrase activity in the context of the HIV-1 preintegration complex. Here, we study the mechanism of action of 11-mer oligonucleotides, which are efficient inhibitors of the catalytic activity of integrase, provided that they are conjugated to a hydrophobic compound, acridine. To understand the mechanism of the conjugate inhibitory action, we used a steady-state fluorescence anisotropy assay, which allowed us to study the stability of the integrase/DNA complex in various conditions. We found that oligonucleotide-acridine conjugates induced the efficient dissociation of preassembled integrase/DNA complexes. The simultaneous presence of both acridine and an oligonucleotidic moiety is required for the inhibitory activity of conjugates. However, the dissociation effect is not dependent on the oligonucleotide sequence. Finally, our results suggest that the conjugates bind directly to integrase within its complex with DNA at a site different from the viral DNA binding site.

Acridines↗

PubMed2004

HIV-1 integrase can process a 3'-end crosslinked substrate.

Integrase of the human immunodeficiency virus type-1 (HIV-1) recognizes specific sequences located in the U3 and U5 regions at the ends of viral DNA. We synthesized DNA duplexes mimicking the U5 region and containing either 2'-aminonucleosides or non-nucleoside 1,3-propanediol insertions at the third and terminal positions and studied their interactions with HIV-1 integrase. Both modifications introduced a local structural distortion in the DNA double helix. Replacement of the terminal nucleosides by corresponding 2'-aminonucleosides had no significant effect on integrase activity. We used an integrase substrate bearing terminal 2'-aminonucleosides in both strands to synthesize a duplex with cross-linked strands. This duplex was then used to determine whether terminal base pair disruption is an obligatory step of retroviral DNA 3'-processing. Processing of the cross-linked analog of the integrase substrate yielded a product of the same length as 3'-processing of the wild-type substrate but the reaction efficiency was lower. Replacement of the third adenosine in the processed strand by a corresponding 2'-aminonucleoside did not affect integrase activity, whereas, its replacement by 1,3-propanediol completely inhibited 3'-processing. Both modifications of the complementary thymidine in the nonprocessed strand increased the initial rate of 3'-processing. The same effect was observed when both nucleosides, at the third position, were replaced by corresponding 2'-aminonucleosides. This indicates that the local duplex distortion facilitated the cleavage of the phosphodiester bond. Thus, a localized destabilization of the third A-T base pair is necessary for efficient 3'-processing, whereas 3'-end-fraying is important but not absolutely required.

Base Pairing↗

PubMed2002

Disruption of HIV-1 integrase-DNA complexes by short 6-oxocytosine-containing oligonucleotides.

We recently found that oligonucleotides containing the 6-oxocytosine heterocyclic base are efficient inhibitors of the HIV-1 integrase in vitro [Brodin, P., et al. (2001) Nucleosides Nucleotides Nucleic Acids 20, 481-486]. In this report, we demonstrate that the inhibition arises from a noncompetitive mechanism in which the modified oligonucleotide attacks the integrase-DNA complex, leading to its active disruption. This conclusion is based on the following results. First, despite the fact that the respective affinities of a 6-oxocytosine-containing oligonucleotide and of its nonmodified counterpart for integrase were identical, only the modified compound inhibited the enzyme activities. Second, DNA binding and UV cross-linking assays indicated that the 6-oxocytosine-containing oligonucleotide prevented the formation of a stable integrase-DNA complex. Third, the kinetics of the dissociation of the integrase-DNA complex were dramatically accelerated in the presence of the modified ODN, whereas the nonmodified counterpart did not influence the dissociation. This mechanism was supported by the ability of the 6-oxocytosine-containing oligonucleotide to inhibit the strand transfer activity of HIV-1 preintegration complexes in vitro. Disruption of integrase-DNA complexes by 6-oxocytosine-containing oligonucleotides constitutes an original mechanism of integration inhibition, therefore suggesting a strategy for searching for inhibitors of the HIV-1 preintegration complexes.

Binding, Competitive↗