Presteady-state analysis of avian sarcoma virus integrase. I. A splicing activity and structure-function implications for cognate site recognition.
Integrase catalyzes insertion of a retroviral genome into the host chromosome. After reverse transcription, integrase binds specifically to the ends of the duplex retroviral DNA, endonucleolytically cleaves two nucleotides from each 3'-end (the processing activity), and inserts these ends into the host DNA (the joining activity) in a concerted manner. In first-turnover experiments with synapsed DNA substrates, we observed a novel splicing activity that resembles an integrase joining reaction but uses unprocessed ends. This splicing reaction showed an initial exponential phase (k(splicing) = 0.02 s(-1)) of product formation and generated products macroscopically indistinguishable from those created by the processing and joining activities, thus bringing into question methods previously used to quantitate these reactions in a time regime where multiple turnovers of the enzyme have occurred. With a presteady-state assay, however, we were able to distinguish between different pathways that led to formation of identical products. Furthermore, the splicing reaction allowed characterization of substrate binding and specificity. Although integrase requires only a 3' hydroxyl with respect to nucleophiles derived from DNA, it specifically favors the cognate sequence CATT as the electrophile. These experimental results support a two-site "switching" model for binding and catalysis of all three integrase activities.
Bao
Kogan K
KK
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, USA.
Skalka
Anna Marie
AM
Wong
Isaac
I
D017398:Alternative Splicing; D001358:Avian Sarcoma Viruses / Q000201:enzymology*; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D011088:DNA Ligases / Q000378:metabolism; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D019426:Integrases / Q000737:chemistry* / Q000502:physiology*; D007700:Kinetics; D015233:Models, Statistical; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013997:Time Factors; D045567:VDJ Recombinases
D004254:DNA Nucleotidyltransferases; D019426:Integrases; D045567:VDJ Recombinases; D011088:DNA Ligases