Rapid Quench-Flow Assay for Detection of dsDNA Induced Inhibition of Enzyme Self-Adenylylation

Rapid quench experiments were performed using a KinTek Rapid Quench Flow (RQF)-3 (Kintek Corporation, Austin TX) instrument. Samples were prepared with 5 μM deadenylylated T4 DNA ligase with and without 5 μM or 50 μM I-75-dsDNA in 1X ATP-free ligase reaction buffer in syringe A and ATP (2 mM added to 1X ATP-free ligase reaction buffer containing 200 μCi of [α-³²P] ATP/mL (Perkin Elmer, Waltham, MA)) solution in syringe B. Drive syringes contained 1X ATP-free ligase reaction buffer, and a quench composed of 250 mM EDTA plus 0.25% SDS was used. Collected time points were treated by passing 50 μL through a Centrisep^® 10 spin column equilibrated in 50 mM Tris pH 7.5, 0.1% SDS. The flow-through was mixed with 8 μL of 6X Blue Gel Loading Dye (New England Biolabs), and 20-μL aliquots were loaded onto (4–20%) Tris Glycine SDS-PAGE gels and run at 120 V for 30 minutes. The gels were Coomassie stained, the protein band was cut out, and the amount of ³²P-AMP incorporation was counted on a Tri-carb 2900TR liquid scintillation counter (Perkin Elmer, Waltham, MA). Maximal enzyme adenylylation was determined by the amount of ³²P detected after a 60-second reaction time point. Reaction progress is reported as the fraction of this maximal adenylylation.

Self-adenylylation single turnover rate was determined under saturating ATP conditions by fitting the data with a single exponential equation (Eq 6):

where A is the reaction amplitude, and k is the observed single turnover rate. Reported experimental data are the average of a minimum of three replicates, and error reported is the standard error of the measurements.