Comparison of DNA binding and integration half-site selection by avian myeloblastosis virus integrase.
Insertion of the linear retrovirus DNA genome into the host DNA by the virus-encoded integrase (IN) is essential for efficient replication. We devised an efficient virus-like DNA plasmid integration assay which mimics the standard oligonucleotide assay for integration. It permitted us to study, by electron microscopy and sequence analysis, insertion of a single long terminal repeat terminus (LTR half-site) of one plasmid into another linearized plasmid. The reaction was catalyzed by purified avian myeloblastosis virus IN in the presence of Mg2+. The recombinant molecules were easily visualized and quantitated by agarose gel electrophoresis. Agarose gel-purified recombinants could be genetically selected by transformation of ligated recombinants into Escherichia coli HB101 cells. Electron microscopy also permitted the identification and localization of IN-DNA complexes on the virus-like substrate in the absence of the joining reaction. Intramolecular and intermolecular DNA looping by IN was visualized. Although IN preferentially bound to AT-rich regions in the absence of the joining reaction, there was a bias towards GC-rich regions for the joining reaction. Alignment of 70 target site sequences 5' of the LTR half-site insertions with 68 target sites previously identified for the concerted insertion of both LTR termini (LTR full-site reaction) indicated similar GC inflection patterns with both insertional events. Comparison of the data suggested that IN recognized only half of the target sequences necessary for integration with the LTR half-site reaction.
Grandgenett
D P
DP
Institute for Molecular Virology, St. Louis University Medical Center, Missouri 63110.
Inman
R B
RB
Vora
A C
AC
Fitzgerald
M L
ML
D009189:Avian Myeloblastosis Virus / Q000201:enzymology*; D001482:Base Composition; D002874:Chromosome Mapping; D003432:Cross-Linking Reagents; D004254:DNA Nucleotidyltransferases / Q000378:metabolism* / Q000648:ultrastructure; D004279:DNA, Viral / Q000378:metabolism* / Q000648:ultrastructure; D004926:Escherichia coli / Q000235:genetics; D019426:Integrases; D008854:Microscopy, Electron; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic; D016662:Virus Integration
D003432:Cross-Linking Reagents; D004279:DNA, Viral; D004254:DNA Nucleotidyltransferases; D019426:Integrases