Protein co-elution by analytical gel filtration alone or coupled to SEC-MALS was carried out using an ÄKTAmicro (GE Healthcare) FPLC at room temperature. Lig PIP mutants incubated with PCNA and DNA were passed over a 3.2/30 Superdex 200 Increase (GE Healthcare) gel filtration column equilibrated in 20 mM MES pH 6.5, 50 mM NaCl, 10 mM MnCl2 and 0.1 mM TCEP. The 10/300 Superdex 200 Increase gel filtration column was used to analyze complexes undergoing DNA ligation (buffer: 25 mM HEPES pH 7.5, 10 mM MnCl2, 50 mM NaCl, 1 mM ATP) and complexes of Lig DBD mutants with PCNA (buffer: 25 mM HEPES pH 7.5, 50 mM NaCl, 10 mM MnCl2, 0.1 mM TCEP).
The Lig-PCNA-DNA molecular mass was measured using the 10/300 Superdex 200 Increase gel filtration column coupled in-line to a UV detector, Dawn HELEOS II MALS flow cell (Wyatt Technology) and OptiLab T-rEX differential refractometer (Wyatt Technology). The buffer contained 20 mM MES pH 6.5, 50 mM NaCl, 10 mM MnCl2 and the detectors were equilibrated with BSA. Data were processed using ASTRA Version 6.1.6.5 (Wyatt Technology).
The Lig-PCNA-DNA molecular mass was measured using the 10/300 Superdex 200 Increase gel filtration column coupled in-line to a UV detector, Dawn HELEOS II MALS flow cell (Wyatt Technology) and OptiLab T-rEX differential refractometer (Wyatt Technology). The buffer contained 20 mM MES pH 6.5, 50 mM NaCl, 10 mM MnCl2 and the detectors were equilibrated with BSA. Data were processed using ASTRA Version 6.1.6.5 (Wyatt Technology).