Document 0712
 DOCN  M9590712
 TI    Coordinated disintegration reactions of M-MuLV integrase.
 DT    9509
 AU    Roth MJ; Donzella GA; UMDNJ-RWJ Medical School, Dept of Biochemistry,
       Piscataway, NJ
 SO    NIH Conf Retroviral Integrase. 1995 Jan 19-20;:(Session II, speakers'
       abstracts - unpaged). Unique Identifier : AIDSLINE AIDS/95920007
 AB    A coordinated disintegration assay (crossbone disintegration) was
       utilized to investigate the possible protein-DNA and protein-protein
       interactions that are important for IN function. The substrate is an
       oligonucleotide which resembles a viral LTR joined to a partial target
       DNA site. Coordination of two oligonucleotides by M-MuLV IN generates an
       integration intermediate, or crossbone structure, and promotes
       intermolecular disintegration at one or two LTR/target junction ends.
       The reaction also yields an intramolecular product (foldback) that
       results from attack of the target 3'-OH on its' own LTR. An IN mutant
       which lacks the HHCC finger (Delta 105) was capable of intermolecular
       disintegration, but was impaired for the intramolecular foldback
       reaction. Another mutant, Delta 177, deletes the N- terminus through the
       first aspartate of the catalytic triad and was incapable of substrate
       coordination. Complementation of Delta 105 with inactive C-terminal
       mutants or a separately purified HHCC finger domain restored the
       intramolecular reaction. In contrast, Delta 177 could not be
       complemented by any mixture of proteins. The roles of LTR and target DNA
       determinants on crossbone substrate coordination were also addressed.
       Prior to integration, IN recesses the 3' end of the LTR substrate,
       leaving a two nucleotide 5' overhang (5'-tail). In the coordinated
       disintegration assay, eliminating this 5'-tail from the crossbone
       substrate LTRs severely impaired the intramolecular foldback reaction,
       but had no effect on intermolecular disintegration. Crossbone substrates
       lacking the 5'-tail were not recognized by the fingerless Delta 105
       protein, suggesting the HHCC finger and LTR 5'-tail possess similar or
       redundant functions. Oligonucleotides representing the target or LTR
       regions of the crossbone structure were both capable of independent
       coordination by M-MuLV IN, and coordination was dependent on presence of
       either an LTR 5' tail or the HHCC finger.
 DE    *DNA Nucleotidyltransferases/*METABOLISM  DNA,
       Viral/*GENETICS/METABOLISM  DNA-Binding Proteins/METABOLISM  Moloney
       Leukemia Virus/*ENZYMOLOGY  Mutation  Oligonucleotides/METABOLISM
       Protein Folding  Repetitive Sequences, Nucleic Acid  Substrate
       Specificity  Virus Integration/*GENETICS  MEETING ABSTRACT

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