DNA repair scheme gets closer look for cancer therapy RiceUniversity
value at each enzyme concentration for each mutant in comparison to the WT. We considered10 nM of HP [3,9] and increasing concentration of WT or mutant LcPolθ-pol were mixed and incubated on ice for 20 min in 20 μl of binding buffer containing 25 mM HEPES [pH 8.0], 100 mM KCl, 3 mM DTT, 100 μg/ml BSA, 10% glycerol, 2.5 mM EDTA and 0.01% NP40. Binding of LcPolθ-pol to HP [3,9] was analyzed by 6% native PAGE buffered with 1× Tris-glycine.
For LcPol θ-pol complexed with the hairpin substrate HP [3,9], data collection was performed in an automatic manner using EPU software . Movies were recorded at defocus values from –1.0 to –2.0 μm at a magnification of 105k×, which corresponds to the pixel size of 0.826 Å at the specimen level . During 3.0-second exposure, 60 frames were collected with a total dose of 67 electrons Å. In total, 4357 images were collected.
Data processing strategies were same for the two datasets. Motion correction was performed on raw super-resolution movie stacks and binned by 2 using MotionCor2 software (
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