Fig. 4: PCNA interaction-dependent modulation of Lig1 ligation rates in the presence of FEN1.

a Schematics of the experiment shown in panel (b). b Multiple-turnover ligation by Lig1 in the presence of trapped PCNA and competitor FEN1. A double-blocked (NeutrAvidin bound to both DNA termini; 1 µM NeutrAvidin) DNA nick substrate (500 nM) was used for this experiment. PCNA (500 nM) was loaded on this substrate by RFC (500 nM). FEN1 D181A (500 nM) was added and incubated with the PCNA-loaded substrate. The reaction was initiated by adding WT_Lig1 or LML_Lig1 (1 nM) and incubated for the indicated amount of time at 37 °C. c Quantification of the data from panel (b) as described in the Methods section. The single-exponential burst parameters are irrelevant kinetically and only their derivative near t = 0 is employed to estimate the normalized initial rate. For WT_Lig1 the burst parameters were A = (183.9 ± 25.2) nM and τ_obs = (311.4 ± 103.5) s. For LML_Lig1 the burst parameters were A = (123.3 ± 38.0) nM and τ_obs = (625.8 ± 324.6) s. The experimental data points represent the mean and one standard deviation of N = 3 independent reactions. d Bar chart showing the initial ligation rates of WT_Lig1 and Lig1 variants quantified from the derivative of the bursts presented in Fig. 4c and Supplementary Fig. 11c near t = 0. The bar chart illustrates the mean (as bar height) and one standard deviation (as error bar) of N = 3 independent reactions. Source data are provided as a Source Data file.