Microscale Thermophoresis.

Thermophoresis is defined as the movement of molecules in response to a temperature gradient, and the response typically changes in response to even small changes in size or charge (41). Thus, a protein complex moves at a different rate than the individual proteins themselves, allowing for calculation of a binding isotherm (Fig. 1A). MST experiments were performed in 1× MST buffer (25 mM Tris⋅HCl pH 7.5, 100 mM K glutamate, 10% glycerol, 5 m DTT, 1 mM EDTA, and 0.05% Tween 20). Before the experiment, a stock of Cy5-labeled CMG or PCNA (Proteins and DNA Constructs) was diluted into this buffer at a ratio of ∼1:20 to ∼8 nM for CMG or ∼1:125 to ∼30 nM for PCNA. Exonuclease deficient Pol ε and Pol δ (Proteins and DNA Constructs) were dialyzed into this buffer for 4 h before the experiment. All proteins were spun at 14,000 rpm for 30 min at 4 °C to remove insoluble aggregates. For CMG binding experiments, Pol ε or Pol δ was added to an equal volume of Cy5-CMG (20 μL final; ∼4 nM CMG probe final) at the indicated concentrations. For PCNA-binding experiments, 30 nM of PCNA-Cy5 was loaded onto 250 nM primed DNA substrate (Proteins and DNA Constructs) with 25 nM RFC, 2.5 mM ΑTP, and 10 mM MgOAc, in 1× MST buffer. RFC was allowed to load the fluorescently labeled PCNA clamp onto the primed DNA for 5 min. Fractions of this reaction were mixed with equal volumes of the indicated concentrations of Pol ε or Pol δ. All reactions were allowed to equilibrate for 10 min at RT and were subsequently drawn into to a surface-passivated glass capillary tube (NanoTemper Technologies). The thermophoresis profile at each concentration point was quantified in a Monolith NT.115 instrument (NanoTemper Technologies) using a 647-nM excitation LED laser set at 30% gain and an IR laser (activated from t = 0 to t = 30) set at 25% power for CMG experiments and 10% power for PCNA experiments. All experiments were performed in triplicate. To determine the dissociation constant of the interactions, MO.Affinity software (NanoTemper) was used to fit the thermophoresis data to a simple mass action equation for a 1:1 binding mode, solving for Kd: