Characterization of the forward and reverse integration reactions of the Moloney murine leukemia virus integrase protein purified from Escherichia coli.
The forward and reverse reactions for integration were characterized for the Moloney murine leukemia virus integrase (M-MuLV IN) protein. The M-MuLV IN was recombinantly produced in Escherichia coli, and was purified to greater than 90% homogeneity by a one-step affinity purification scheme. M-MuLV IN was highly active for integration as measured by in vitro cleavage and strand transfer assays. Furthermore, the integration of a model viral substrate into lambda concatamers by IN correctly produced the flanking 4-base pair duplications characteristic of M-MuLV IN. The reverse reaction of integration, disintegration, was also catalyzed by the recombinant M-MuLV IN. Two products were generated, a 3'-recessed long terminal repeat and a ligated target DNA, from a model integration-intermediate substrate in the presence of M-MuLV IN. The requirements and optimal conditions for maximal integration and disintegration activity for M-MuLV IN were determined. The forward and reverse reactions required different concentrations of manganese ion and reductant. Salt was also titrated for the forward and reverse reactions. Sodium chloride inhibited integration, but had little affect on disintegration. Low concentrations of potassium chloride enhanced integration, but had no affect on disintegration. The dinucleotide cleavage, strand transfer, and the disintegration reactions each had a unique pH profile of activity.
Jonsson
C B
CB
Department of Biochemistry, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway 08854.
Donzella
G A
GA
Roth
M J
MJ
D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005822:Genetic Vectors; D019426:Integrases; D007700:Kinetics; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000201:enzymology* / Q000235:genetics; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004254:DNA Nucleotidyltransferases; D019426:Integrases