       Document 0043
 DOCN  M9610043
 TI    Characterization of endonucleolytic activity of HIV-1 integrase using a
       fluorogenic substrate.
 DT    9601
 AU    Lee SP; Censullo ML; Kim HG; Knutson JR; Han MK; Department of
       Biochemistry and Molecular Biology, Georgetown; University Medical
       Center, Washington, DC 20007, USA.
 SO    Anal Biochem. 1995 May 20;227(2):295-301. Unique Identifier : AIDSLINE
       MED/96023410
 AB    Retroviruses require viral DNA to be synthesized by reverse
       transcription in the cytoplasm followed by integration of the resulting
       viral DNA into the host chromosome in the nucleus. Reverse transcription
       and integration, essential steps in the life cycle of retroviruses, are
       possible targets in the development of antiviral reagents. One
       attractive target is the integrase protein, a product of the retroviral
       pol gene which is solely responsible for the retroviral integration
       process through cutting and joining reactions. When screening for
       massive numbers of antiviral agents, a rapid and precise assay is ideal.
       We report the application of fluorescence resonance energy transfer
       (FRET) with fluorescein and eosin as the energy transfer pair to
       characterize HIV-IN-mediated DNA cleavage reactions. Past concerns with
       applications of FRET to DNA were due to interactions of the fluorophore
       with the DNA, resulting in quenched fluorescence. However, in this study
       these concerns have been resolved with the use of a nucleotide analog
       with a 12-carbon linker arm, 5-amino (12)-2'-deoxyuridine
       beta-cyanoethyl phosphoramidite. Steady-state fluorescence studies show
       that cleavage of the fluorogenic substrate by integrase results in
       enhancement of quenched donor fluorescence intensity. The fluorescence
       assay was confirmed by autoradiographic analysis of the cleavage
       reaction with radiolabeled fluorogenic substrate. This fluorescence
       assay will facilitate both detailed kinetic studies and the rapid
       screening of novel integrase inhibitors.
 DE    Base Sequence  Deoxyuridine/ANALOGS & DERIVATIVES  DNA/CHEMISTRY
       Endodeoxyribonucleases/*ANALYSIS  Energy Transfer  Eosine
       Yellowish-(YS)/*CHEMISTRY  Fluoresceins/*CHEMISTRY  Fluorescent Dyes
       Molecular Sequence Data  Spectrometry, Fluorescence  Support, Non-U.S.
       Gov't  JOURNAL ARTICLE

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

