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 realtime. Because the initial anisotropy value obtained after DNA binding and before catalysis depends on the fractional saturation of DNA sites and the size of IN⅐DNA 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.The integration of a DNA copy of the HIV-1 2 genome into the host genome is a crucial step in the life cycle of the retrovirus. Integrase (IN) is responsible for the two consecutive reactions that constitute the overall integration process. The first of these two reactions is 3Ј-processing, which involves cleavage of the 3Ј-terminal GT dinucleotide at each extremity of the viral DNA. The hydroxyl groups of newly recessed 3Ј-ends are then used in the second reaction, strand transfer, for the covalent joining of viral and target DNAs, resulting in full-site integration. IN is sufficient for catalysis of the 3Ј-processing reaction in vitro, using short-length oligodeoxynucleotides (ODNs) that mimic one viral long terminal repeat (LTR) in the presence of the metallic cofactor Mg 2ϩ . This reaction generates two products: the viral DNA containing the recessed extremity and the GT dinucleotide. One of the two products, the processed viral DNA, as well as the target DNA serve as substrates for the subsequent joining reaction.IN belongs to the superfamily of polynucleotidyl transferases. Its catalytic core domain contains a triad of acidic residues constituting the D,D-35-E motif, which is strictly required for catalysis. The catalytic core establishes specific contacts with the viral DNA and, together with the C-terminal domain, is involved in DNA binding (1-4). ...

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 Mn 2؉ or Mg 2؉ 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 Mg 2؉ than in the presence of Mn 2؉ . 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.Following reverse transcription, a DNA copy of the human immunodeficiency virus, type 1 (HIV-1), 2 RNA is integrated into the genome of infected cells. Integration is a prerequisite for viral replication and is catalyzed by the viral enzyme integrase (IN). IN binds to sequences located at the end of U3 and U5 parts of long terminal repeats (LTRs) of viral DNA and catalyzes the trimming, or 3Ј-end processing, of the terminal dinucleotide from the 3Ј-ends of both strands of the DNA. IN then mediates a strand transfer reaction that inserts the viral DNA into the host DNA. During this reaction, IN must bind simultaneously to viral and target DNA. However, IN interacts with these two DNA molecules in different ways as follows: binding to host DNA does not depend directly on host DNA sequence, whereas interaction with the viral DNA is a sequence-specific process. Nevertheless, the U5 and U3 sequences recognized by IN are not exactly identical.Strand transfer and 3Ј-end processing reactions may be carried out in vitro, using recombinant HIV IN, DNA duplexes mimicking U3 or U5 sequences of LTRs, and divalent metal ions, such as Mg 2ϩ or Mn 2ϩ . However, the Mn 2ϩ -and Mg 2ϩ -dependent activities of IN are not equivalent, with lower specificity reported for Mn 2ϩ -dependent IN (1, 2). Moreover, the inhibition of HIV-1 IN by compounds such as ␤-diketo acids, which interact with the active site of HIV-1 IN, is also metal-de...

The published data on the methods of chemical solution The published data on the methods of chemical solution and solid-phase synthesis of peptide ± oligonucleotide conjugates and solid-phase synthesis of peptide ± oligonucleotide conjugates are reviewed. The known methods are systematised and their are reviewed. The known methods are systematised and their advantages and disadvantages are considered. The approaches to advantages and disadvantages are considered. The approaches to the solution synthesis of peptide ± oligonucleotide conjugates are the solution synthesis of peptide ± oligonucleotide conjugates are systematised according to the type of chemical bonds between the systematised according to the type of chemical bonds between the fragments, whereas those to the solid-phase synthesis are classified fragments, whereas those to the solid-phase synthesis are classified according to the procedure used for the preparation of conjugates, according to the procedure used for the preparation of conjugates, viz viz., stepwise elongation of oligonucleotide and peptide chains on ., stepwise elongation of oligonucleotide and peptide chains on the same polymeric support or solid-phase condensation of two the same polymeric support or solid-phase condensation of two presynthesised fragments. The bibliography includes 141 referen-presynthesised fragments. The bibliography includes 141 references ces. .

An efficient method for synthesis of oligonucleotide 2′-conjugates via amide bond formation on solid phase is described. Protected oligonucleotides containing a 2′-O-carboxymethyl group were obtained by use of a novel uridine 3′-phosphoramidite, where the carboxylic acid moiety was introduced as its allyl ester. This protecting group is stable to the conditions used in solid-phase oligonucleotide assembly, but easily removed by Pd(0) and morpholine treatment. 2′-OCarboxymethylated oligonucleotides were then efficiently conjugated on a solid support under normal peptide coupling conditions to various amines or to the N-termini of small peptides to give products of high purity in good yield. The method is well suited in principle for the preparation of peptide-oligonucleotide conjugates containing an amide linkage between the 2′-position of an oligonucleotide and the N-terminus of a peptide.

Oligonucleotide-peptide conjugates have several applications, including their potential use as improved antisense agents for interfering with the RNA function within cells. In order to provide robust and generally applicable conjugation chemistry, we developed a novel approach of fragment coupling of pre-synthesized peptides to the 2P P-position of a selected nucleotide within an otherwise protected oligonucleotide chain attached to a solid support.z 1999 Federation of European Biochemical Societies.