Rapid solution assays for retroviral integration reactions and their use in kinetic analyses of wild-type and mutant Rous sarcoma virus integrases.
A rapid method for quantitating products of the oligodeoxynucleotide processing reaction in vitro has been developed to facilitate enzymatic studies of the retroviral integrases. Unlike earlier procedures, this assay does not depend on polyacrylamide gel electrphoresis but separates products by batch adsorption to PEI-cellulose. A joining assay has also been modified, to facilitate measurement of the two distinct steps in the integration reaction under parallel conditions. Since these methods allow quantitation of numerous samples in a short period of time, they are especially useful for investigation of kinetic parameters and to measure the effects of possible inhibitors of integrase. These assay systems were used to examine the enzymatic activity of wild-type Rous sarcoma virus integrase and selected mutant proteins with substitutions of single conserved amino acids. In contrast to previous studies, reactions were performed under conditions of substrate excess, and rates, rather than yields of product generated after a given period of incubation, were determined. The results showed that substitutions of several highly conserved residues in what is most likely an evolutionarily conserved catalytic domain of the integrases resulted in a 4- to 10-fold decrease in the apparent rate of processing relative to wild type, under optimized standard conditions. Changing an invariant acidic residue reduced the rate by approximately 60-fold. When joining activity was determined, the relative effects of the substitutions tested generally paralleled the results with processing. However, with both wild-type and mutant integrase proteins, the linear phase of the joining reaction was preceded by what appears to be an exponential "burst" phase.
Müller
B
B
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111.
Jones
K S
KS
Merkel
G W
GW
Skalka
A M
AM
D000595:Amino Acid Sequence; D001358:Avian Sarcoma Viruses / Q000201:enzymology* / Q000235:genetics*; D002852:Chromatography, Ion Exchange; D017124:Conserved Sequence; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D019426:Integrases; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D016662:Virus Integration
D009838:Oligodeoxyribonucleotides; D004254:DNA Nucleotidyltransferases; D019426:Integrases