Document 0713
 DOCN  M9590713
 TI    Processing of modified DNA substrates by HIV-1 integrase.
 DT    9509
 AU    Mazumder A; Pommier Y; Laboratory of Molecular Pharmacology, National
       Cancer Insitutite,; NIH, Bethesda, MD
 SO    NIH Conf Retroviral Integrase. 1995 Jan 19-20;:(Session II, speakers'
       abstracts - unpaged). Unique Identifier : AIDSLINE AIDS/95920006
 AB    The extents of strand transfer and 3'-processing via hydrolysis,
       alcoholysis, and circular nucleotide formation were examined using
       backbone-modified substrates. These substrates had a negatively charged
       phosphodiester linkage replaced by a neutral, hydrophobic
       methylphosphonodiester at various positions near the conserved CA
       dinucleotide. Substitution of the phosphodiester 3' to the scissile
       phosphodiester was found to block 3'-processing and to alter the choice
       of nucleophile. Nucleophilic attack by water and glycerol was blocked
       while that by the viral DNA hydroxyl end was stimulated. These effects
       could reflect disruption of electrostatic interactions or introduction
       of steric hinderance in the interaction of the substrate with the
       enzyme. Mutagenesis of the enzyme active site has previously been shown
       to alter the choice of nucleophile in the 3'- processing reaction. Taken
       together, our results suggest that mutagenesis of the DNA backbone can
       also alter the choice of nucleophile. We have also examined processing
       of substrates containing deoxyuridine (dU) mismatches or abasic (AP)
       sites within and near the conserved CA dinucleotide of the U5 end of the
       HIV-1 LTR. Although DNA binding was not affected, dU substitution in the
       processed strand of either the C or A of the CA dinucleotide or of the G
       5' to the CA reduced strand transfer six-, three-, and seven-fold,
       respectively. Substitution in the nonprocessed strand of either the G or
       T nucleotide complementary to the C and A nucleotides, respectively,
       abolished strand transfer in the case of the G but had minimal effect in
       the case of the T. Therefore, the G, C, and A are critical for efficient
       strand transfer by HIV-1 integrase. 3' processing was only reduced by
       substitution at the GC. Substitution outside this trinucleotide remained
       compatible with enzyme activity. When an AP site was substituted for
       either of the nucleotides of the GCA trinucleotide, 3' processing and
       strand transfer were abolished. Using these oligonucleotides, we found a
       yet undescribed AP endonuclease activity, resulting in nicking 3' to the
       AP site, even with single-stranded or random duplex AP substrates. The
       integrase D116N mutant nicked linear AP substrates. Taken together,
       these results suggest that base-mismatched or base-deleted substrates,
       which can be created by the proofreading-deficient HIV-1 RT, can be
       processed by HIV-1 integrase.
 DE    DNA Nucleotidyltransferases/*METABOLISM  DNA,
       Viral/CHEMISTRY/*GENETICS/METABOLISM  Electrochemistry
       HIV-1/*ENZYMOLOGY  Hydrolysis  Mutagenesis  *Protein Processing,
       Post-Translational  Substrate Specificity  MEETING ABSTRACT

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