Fig. 3: Biochemical assays of Lig1 binding to PCNA in absence and presence of DNA.

a Schematics of the experiment shown in panel (b). b Protein–protein EMSA was used to monitor the binding of WT_Lig1 and LML_Lig1 to Cy5-labeled PCNA (1 nM) in solution in the absence of DNA. c Quantification of the EMSA data presented in panel (b). The experimental data points were fitted to quadratic dependencies as described in “Methods”. d Schematics of the experiment shown in panel (e). e Emission spectra of DNA-Alexa Fluor 647 and PCNA-Cy3 in the absence and presence of WT_Lig1 or Lig1 variants. DNA-Alexa Fluor 647 (500 nM) was blocked with NeutrAvidin (1 μM) at the end of the downstream arm to prevent PCNA sliding-in from that end and to allow for PCNA sliding-in only through the 3’ duplex arm. The nicked DNA contained a 3’ ddC to prevent ligation. PCNA-Cy3 (500 nM; 3:1 = Cy3:PCNA) was then added. The direct sum (‡) of PCNA-Cy3 and DNA-Alexa Fluor 647 fluorescence emission intensity (upon excitation at 480 nm) is used as a baseline. The inset shows a closer view of the Alexa Fluor 647 emission band. f Quantification of the data presented in panel e. The emission spectra of the baseline (‡), PCNA-Cy3-DNA-Alexa Fluor 647 in the absence of Lig1 and in the presence of WT_Lig1 or Lig1 variants were fitted with a linear combination of Cy3 and Alexa647 emission spectra. The deconvoluted amplitude of the Alexa Fluor 647 emission contribution was recorded for each of the four spectra. The value corresponding to the baseline condition (‡) was subtracted from the other six conditions. The bar chart shows the quantified increase in Alexa Fluor 647 emission, above the baseline, for the six conditions. The bar chart illustrates the mean (as bar height) and one standard deviation (as error bar) of N = 3 independent measurements. Source data are provided as a Source Data file.