Document 0710
 DOCN  M9590710
 TI    Analysis of in vitro hiv-1 dna integration reaction by uv cross-linking
 DT    9509
 AU    Fujiwara T; Yoshinaga T; Shionogi Institute for Medical Science,
       Shionogi & Co, Ltd.
 SO    NIH Conf Retroviral Integrase. 1995 Jan 19-20;:(Session II, speakers'
       abstracts - unpaged). Unique Identifier : AIDSLINE AIDS/95920009
 AB    The DNA sequence requirements for the retroviral DNA integration
       reaction suggest the existence of a sequence- specific binding of
       integrase (IN) with substrate DNA. We detected three photoadduct bands
       applying UV cross-linking method to an integration reaction mixture
       consisted of recombinant HIV-IN and oligonucleotide substrate.
       Investigation of these photoadduct bands with (1) wild type, base
       substituted, deleted, and mismatched mutant substrates, (2) time course
       experiment, (3) substrates labeled at different positions, (4)
       N-ethylmaleimide, and (5) integration reaction after UV cross-linking,
       proved that we detected a functional complex of HIV-1 IN and its DNA
       substrates. Our results also suggest that some bases of the HIV-1 LTR
       are primarily required for binding, whereas others are more critical for
       later reaction steps. On the basis of these observations, we propose
       that an integration reaction can be divided as follows. First, IN binds
       the substrate DNA independent of sequence but may preferentially
       recognize a DNA end structure (an initial binding complex). Second, IN
       recognizes the sequence of terminal thirteen base pairs of LTR and binds
       the substrate DNA sequence specifically prior to 3' processing (a
       precreavage complex). The terminal two nucluotides of the U5 plus strand
       are important for this binding. The time necessary from the initial
       binding step to the 3' step process may be longer than the following
       strand transfer step. The terminal part of each strand of both ends of
       the viral DNA may be separated during the 3'processing and strand
       transfer steps. Terminal three nucleotides of the U5 minus strand may be
       important for the joining activity by playing some role for this strand
       separation. Target DNA probably binds to another site of IN at some time
       before the DNA strand transfer reaction. By the completion of the strand
       transfer, IN dissociates from the substrate. We also propose to
       designate the terminal six base pairs of HIV-1 LTR necessary for almost
       full activity as the integration signal sequence (ISS).
 DE    DNA Nucleotidyltransferases/*METABOLISM  DNA, Viral/*GENETICS/METABOLISM
       Ethylmaleimide  HIV Long Terminal Repeat  HIV-1/ENZYMOLOGY/*GENETICS
       Protein Processing, Post-Translational  Recombinant Proteins/METABOLISM
       Ultraviolet Rays  Virus Integration/*GENETICS  MEETING ABSTRACT

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