Document 0691
 DOCN  M9590691
 TI    Oligonucleotide Binding Properties of the HIV-1 Integrase - A Potential
       to wrap single stranded DNA?
 DT    9509
 AU    Pemberton I; Buckle M; Buc H; Unite de Physicochemie des Macromolecules
       Biologique, Institut; Pasteur, Paris
 SO    NIH Conf Retroviral Integrase. 1995 Jan 19-20;:(Participants' abstracts
       and posters, abstract no. 7). Unique Identifier : AIDSLINE AIDS/95920028
 AB    HIV-1 IN performs the catalytic functions required for the integration
       of a linear DNA molecule provided that authentic viral LTR sequences are
       present at both DNA termini. Apparently anomolously, however, in vitro
       the HIV-1 IN binds to non-viral DNA substrates with an affinity
       comparable to that observed for sequences derived from the viral LTRs.
       Furthermore, single strand DNA, for which the HIV-1 IN displays no
       catalytic activity, is an equally efficient substrate for DNA-binding.
       In order to explore the nucleic acid binding properties of the HIV-1 IN
       more fully, we have exploited the ability of rapid pulse (nanosecond)
       laser- mediated UV cross-linking to capture, in quantum yield, a
       representation of the binding equilibria in the form of zero length
       covalent protein-DNA adducts. The yield of adduct formation is both a
       function of the fraction of potential binding sites occupied and the
       photochemical reactivity of the bound DNA sequence. Thus, binding
       affinities are measured by titrating the free DNA sites with increasing
       concentrations of the protein ligand and reporting these in terms of
       fractional saturation. By this approach we have made the following
       observations. (1) No sequence selectivity is observed in terms of
       apparent Kd (1-2 x 10(- 7) M) for the binding of single (ss) or double
       (ds) strand oligonucleotides. (2) Non-specific binding to short ss
       oligonucleotides is subject to rapid equilibrium with a dissociation
       rate typically in the order of 1 sec-1 (for a 21mer) and may be
       perturbed equally before or after the formation of complexes by NaCl or
       DNA competition. (3) Irrespective of length (between 10 and 100
       nucleotides) or sequence, at saturation all bases on a ssDNA molecule
       may be occluded by the HIV-1 IN in the context of an intimate
       nucleoprotein complex and each base may participate equally in the
       formation of adducts. (4) Detailed analysis of the protein to DNA ratio
       of such complexes suggests that the stoichiometric number of apparent
       binding sites increases with the progressive saturation of the DNA
       (i.e., the binding site size per monomer appears to decrease), possibly
       as a result of protein:protein interactions that preclude protein:DNA
       interactions. Such alterations in stoichiometry are not consistent
       however with a simple aggregation of the IN in solution, but rather are
       linked to the degree of DNA lattice saturation, as evidenced when
       titrations are conducted over a wide range of DNA concentrations.
       Fluctuations in the apparent binding density appear to be constrained by
       the free, rather than the total, HIV-1 IN concentration. (5) At half
       saturation, a value of 18 nucleotides occluded per monomer is indicated
       for poly d(T). Combined, these data suggest that the HIV-1 IN may wrap,
       or otherwise bind, ssDNA contiguously in the form of a high order
       multimeric nucleoprotein complex. The structural architecture of such
       complexes remains to be established.
 DE    *Chromosomes, Fungal  Cloning, Molecular  DNA Repair  DNA Replication
       DNA, Complementary  Human  Mutation  Promoter Regions (Genetics)
       Recombination, Genetic  Saccharomyces cerevisiae/*GROWTH &
       DEVELOPMENT/GENETICS  Sequence Deletion  MEETING ABSTRACT

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).