Mars software package

Helicase activity was analyzed utilizing a fluorescence-based assay. We used an open fork substrate with a Cy3 label at the 3′ end of the translocated strand where unwinding of the DNA substrate reduces quenching of the Cy3 fluorescence.
Assays were carried out in 20 mM HEPES pH 7.5, 50 mM KCl, 5 mM MgCl₂, and 1 mM TCEP. DNA was used at a concentration of 250 nM. Helicase activity was measured with equimolar concentrations of XPD in the presence of N-p44 or p44/p62, respectively. The mix of reagents, were preincubated at 37°C and the reaction was subsequently started with the addition of 5 mM ATP. Kinetics were recorded with a Fluostar Optima plate reader (BMG labtech). Fluorescence was detected at an excitation wavelength of 550 nm (slit width, 2 nm) and an emission wavelength of 570 nm (slit width, 2 nm). Initial velocities were fitted with the MARS software package (BMG labtech) and represent the averages of at least three different reactions and two independent protein batches.

Kinase activity was determined using an in vitro ATPase assay in which ATP consumption is coupled to the oxidation of NADH via pyruvate kinase and lactate dehydrogenase activities. Kinase activities were assessed at 37 °C, containing 1.5 U pyruvate kinase, 1.9 U lactate dehydrogenase, 2 mM phosphoenolpyruvate, 0.3 mM NADH, 20 mM Hepes (pH 7.5), 200 mM NaCl, 1 mM TCEP, and 5 mM MgCl₂. The reactions were performed with 200 nM ctCDK7 or ctCDK7-containing complexes and 20 µM MBP-scCTD as the substrate. The mixture including all above-mentioned components was preincubated at 37 °C until a stable baseline was achieved. The reaction was then started by the addition of ATP at a concentration of 1 mM. Activity profiles were recorded at 340 nm using a FluoStar Optima (BMG Labtech) plate reader until NADH was entirely consumed. The velocity increased during the reaction and the highest velocity toward the end of each reaction was fitted with the MARS software package (BMG Labtech). The rate of ATP consumption was calculated using the molar extinction coefficient of NADH (6,220 M⁻¹⋅cm⁻¹). Measurements were carried out in at least triplicate derived from two biological replicates and mean values were plotted with their associated SD.