Document 0698
 DOCN  M9590698
 TI    Real time assay for the hiv-1 integrase 3'-processing reaction using a
       novel fluorescent guanosine analog.
 DT    9509
 AU    Hawkins ME; Pfleiderer W; Balis FM; Pediatric Branch, National Cancer
       Institute, Bethesda, MD
 SO    NIH Conf Retroviral Integrase. 1995 Jan 19-20;:(Participants' abstracts
       and posters, abstract no. 2). Unique Identifier : AIDSLINE AIDS/95920021
 AB    Fluorescence is an important tool for studying protein/DNA interactions
       because fluorophores are sensitive to changes in their molecular
       environment and reflect those changes through measurable differences in
       fluorescence. Unfortunately, most fluorophores are large molecules and,
       when linked to DNA, their presence can change the tertiary structure of
       DNA or interfere with binding and mobility. We have developed a highly
       fluorescent pteridine, 3-methyl-8- (2-deoxy beta-D-ribofuranosyl)
       isoxanthopterin (3-MI), which is a structural analog of guanosine. In
       its phosphoramidite form, 3-MI can be site-specifically inserted into an
       oligonucleotide using an automated DNA synthesizer. Because 3-MI is
       inserted into the DNA through a 3',5'-phosphodiester linkage identical
       to that of naturally occurring nucleotides, it is less likely to alter
       the conformation of DNA or interfere with protein/DNA interactions than
       fluorophores which are linked externally to DNA. Insertion of 3-MI into
       an oligonucleotide partially quenches its fluorescence intensity. This
       new fluorophore was used to develop a real time assay for monitoring the
       HIV-1 integrase 3'processing reaction. Purified recombinant enzyme is
       incumbated with a short oligonucleotide (21-mer) with a sequence
       identical to the U5 terminus of HIV-1 DNA with 3-MI substituted for
       guanosine at the cleavage site. When the 3'-terminal dinucleotide
       containing 3-MI is cleaved by integrase, fluorescence intensity
       increases at a rate proportional to the rate of the reaction. The change
       in fluorescence can be monitored in real time by performing the reaction
       in a spectrofluorometer. Because the change in fluorescence intensity is
       dependent on release of the 3-MI- containing dinucleotide, this assay is
       specific for the 3'- processing reaction. Using this assay, we studied
       the role of cofactor and substrate concentration on the rate of the
       3'-processing reaction, as well as the effect of the chelating agent,
       EDTA, which binds the required divalent cation, Mn2+. In the absence of
       M2+ there is no change in fluorescence intensity confirming that it is a
       required cofactor. The reaction did not proceed unless the Mn2+ was
       added to the reaction mixture (containing the oligonucleotide substrate)
       before integrase, suggesting that integrase must interact with Mn2+
       before binding to the DNA substrate. The optimal Mn2+ concentration in
       our assay system was 7.5-15 nM. Pre-incubation of the enzyme with EDTA
       prior to the addition of enzyme to the reaction mixture completely
       inhibits the reaction, but pre-incubation of the reactants with EDTA
       prior to the addition of enzyme did not immediately inhibit the
       reaction, presumably because of Mn2+ bound to integrase in the enzyme
       preparation. The minimum EDTA concentration necessary to stop a reaction
       which is in progress is 8.0 nM in the presence of 7.5 mM Mn2+. However,
       EDTA added to a reaction in progress does not stop the reaction
       immediately. With increasing substrate concentration, there was evidence
       of substrate inhibition at concentrations exceeding 164 nM. This novel
       real time assay for integrase should prove useful for further
       mechanistic studies of the enzyme and may provide a rapid means of
       screening for inhibitors, which could have therapeutic applications.
 DE    Cations, Divalent  DNA Nucleotidyltransferases/*METABOLISM  DNA,
       Viral/GENETICS  Edetic Acid/CHEMISTRY  Fluorescent Dyes/*CHEMISTRY
       Guanosine/*CHEMISTRY  HIV-1/*ENZYMOLOGY/GENETICS  Manganese/CHEMISTRY
       Protein Processing, Post-Translational  Spectrometry, Fluorescence  Time
       Factors  MEETING ABSTRACT

       SOURCE: National Library of Medicine.  NOTICE: This material may be
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