       Document 0624
 DOCN  M9620624
 TI    Enhanced and coordinated processing of synapsed viral DNA ends by
       retroviral integrases in vitro.
 DT    9602
 AU    Kukolj G; Skalka AM; Institute for Cancer Research, Fox Chase Cancer
       Center,; Philadelphia, Pennsylvania 19111, USA.
 SO    Genes Dev. 1995 Oct 15;9(20):2556-67. Unique Identifier : AIDSLINE
       MED/96033805
 AB    We have designed novel substrates to investigate the first step in
       retroviral integration: the site-specific processing of two nucleotides
       from the 3' ends of viral DNA. The substrates consist of short duplex
       oligodeoxynucleotides whose sequences match those of the U3 and U5 ends
       of viral DNA but are covalently synapsed across the termini by short,
       single-strand nucleotide linkers. We show here that the optimal
       separation between termini in a synapsed-end substrate for avian
       sarcoma/leukosis virus (ASV) IN is 2 nucleotides. This places the two
       conserved 5'-CA-3' processing sites 6 nucleotides apart, a separation
       equal to the staggered cut in target DNA produced by this enzyme during
       the subsequent joining reaction. Based on estimates of initial reaction
       rates, this synapsed-end substrate is processed by IN at > 10-fold
       higher efficiency than observed with an equivalent mixture of U3 and U5
       single-end (uncoupled) substrates. Enhanced processing is maintained at
       low IN concentrations, suggesting that the synapsed-end substrate may
       facilitate enzyme multimerization. Enhanced processing by HIV-1 IN,
       which produces a 5-bp stagger during integration, was observed with a
       synapsed-end substrate in which the separation between processing sites
       was 5 nucleotides. These observations provide estimates of the distances
       between active sites in the multimeric IN-DNA complexes of ASV and
       HIV-1. Our results also show that processing of paired U3 and U5 ends
       need not be coupled temporally. Finally, we observed that substrates
       that paired a wild-type with a mutated terminus were cleaved poorly at
       both ends. Thus, in vitro processing of the synapsed-end substrates
       requires specific recognition of the sequences at both ends. These
       findings provide new insights into the mechanism of integrative
       recombination by retroviral integrases and, by extension, other
       prokaryotic and eukaryotic transposases that are related to the viral
       enzymes.
 DE    Base Sequence  DNA Nucleotidyltransferases/*METABOLISM  DNA,
       Viral/CHEMISTRY/*METABOLISM  HIV-1/*ENZYMOLOGY/GENETICS  Molecular
       Sequence Data  Nucleic Acid Conformation  Sarcoma Viruses,
       Avian/*ENZYMOLOGY/GENETICS  Substrate Specificity  Support, Non-U.S.
       Gov't  Support, U.S. Gov't, P.H.S.  Virus Integration  JOURNAL ARTICLE

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

