ATP hydrolysis is required for DNA cleavage by EcoPI restriction enzyme.
The type III restriction endonuclease EcoPI, coded by bacteriophage P1, cleaves unmodified DNA in the presence of ATP and magnesium ions. We show that purified EcoPI restriction enzyme fails to cleave DNA in the presence of non-hydrolyzable ATP analogs. More importantly, this study demonstrates that EcoPI restriction enzyme has an inherent ATPase activity, and ATP hydrolysis is necessary for DNA cleavage. Furthermore, we show that the progress curve of the reaction with EcoPI restriction enzyme exhibits a lag which is dependent on the enzyme concentration. Kinetic analysis of the progress curves of the reaction suggest slow transitions that can occur during the reaction, characteristic of hysteretic enzymes. The role of ATP in the cleavage mechanism of type III restriction enzymes is discussed.
Saha
S
S
Department of Biochemistry, Indian Institute of Science, Bangalore.
Rao
D N
DN
D000255:Adenosine Triphosphate / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D015263:Deoxyribonucleases, Type III Site-Specific / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D010957:Plasmids
D000255:Adenosine Triphosphate; D004247:DNA; C093225:DNA restriction enzyme EcoPI; D015263:Deoxyribonucleases, Type III Site-Specific