Endonucleolytic cleavages and DNA-joining activities of the integration protein of human foamy virus.
The bacterial expression plasmids, pET3b and pET16b, that contain the integrase domain of the human foamy virus (HFV) reverse transcriptase were constructed and expressed in Escherichia coli. The histidine-tagged HFV IN protein was purified to near homogeneity by single-step Ni2+ chelate affinity chromatography. HFV-specific proteins of 39 and 120 kDa from virus-infected cells reacted with antisera raised against the recombinant IN protein. Purified recombinant HFV IN protein was active as an endonuclease specifically cleaving two nucleotides from a 20-bp oligodeoxynucleotide substrate that mimics the authentic 5' ends of HFV DNA. Substrates with mutations relatively close to the cleavage site were less efficiently cleaved or not cleaved at all compared with the HFV U5 DNA end. The purified recombinant protein was active as integrase with double-stranded oligodeoxynucleotide substrates. The reverse reaction of DNA strand transfer, the disintegration activity, was shown by efficient cleavage of an intermediate Y-shaped oligodeoxynucleotide. In the presence of Mn2+ as the preferred divalent cation, oligodeoxynucleotides were specifically and efficiently cleaved. In contrast, endonucleolytic cleavages in the presence of Mg2+ ions led to a broad range of reaction products with the His-tagged HFV IN protein. After further purification of the HFV IN by cation-exchange chromatography, the unspecific degradation of oligonucleotide substrate in the presence of Mg2+ was not detectable.
Pahl
A
A
Abteilung Retrovirale Genexpression, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Flügel
R M
RM
D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D019426:Integrases; D007700:Kinetics; D008168:Lung; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000138:chemical synthesis; D016133:Polymerase Chain Reaction; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016092:Spumavirus / Q000201:enzymology*; D013379:Substrate Specificity
D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D016376:Oligonucleotides, Antisense; D011994:Recombinant Proteins; D004254:DNA Nucleotidyltransferases; D019426:Integrases; D012194:RNA-Directed DNA Polymerase; D003850:Deoxyribonuclease I