LlaGI Endonuclease Cleavage Kinetics

LlaGI was purified as previously described (18 (link)). A modified version of pRMA03 (18 (link)) was used as a substrate. pRMA03 was digested using SmaI and PshAI and re-ligated generating pRMA03S, a substrate in which the two LlaGI sites are in head-to-head orientation separated by 997 bp. A reaction mix containing 2 nM pRMA03S, 200 nM LlaGI in TMD Buffer (50 mM Tris–Cl, pH 8.0, 10 mM MgCl₂, 1 mM DTT) was pre-incubated at 25°C for 5 min. Cleavage reactions were initiated by the addition of 4 mM ATP, and stopped at 10, 30 or 60 s by adding binding buffer from the DNA Clean & Concentrator-25 kit (Zymo Research) supplemented with 30 mM EDTA and 37 mM sodium acetate. Purification of cleaved DNA proceeded as described by the manufacturer.