Sequence-specific binding of DNA by the Moloney murine leukemia virus integrase protein.
Genetic studies have indicated that integration of retroviral DNA into the host genome depends on the presence of the inverted repeats at the free termini of the long terminal repeats on the unintegrated DNA and on the product of the 3' end of the pol gene (the integrase [IN] protein). While the precise function of the Moloney murine leukemia virus IN protein is uncertain, others have shown that it is a DNA-binding protein and functions in the processing of the inverted repeats prior to integration. By using site-directed mutagenesis, we cloned and expressed the IN protein in Escherichia coli. Crude extracts of total cellular protein were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose filters, denatured in guanidine, renatured, and incubated with oligonucleotide probes. Single- and double-stranded oligonucleotides corresponding to the termini of unintegrated linear viral DNA were specifically bound by the IN protein in this assay. These data suggest that the role of the Moloney IN protein in the early steps of integration involves sequence-specific recognition of the DNA sequences found at the ends of the long terminal repeats.
Krogstad
P A
PA
Department of Pediatrics, University of Washington, Seattle 98195.
Champoux
J J
JJ
D001483:Base Sequence; D003001:Cloning, Molecular; D004254:DNA Nucleotidyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015151:Immunoblotting; D019426:Integrases; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000201:enzymology / Q000235:genetics*; D009154:Mutation; D015345:Oligonucleotide Probes; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D004254:DNA Nucleotidyltransferases; D019426:Integrases