Figure 4: In vivo characterization of UvrB.
From: Single-molecule imaging of UvrA and UvrB recruitment to DNA lesions in living Escherichia coli
(a) Distribution of D* values of 9,958 tracked UvrB molecules, fitted with a three species model (constrained Dimm=0.11 μm2 s−1) established that 15%±2% of UvrB molecules were immobile, 24%±3%, diffusing slowly (Dslow=0.41±0.02 μm2 s−1) and 61%±5% fast diffusing (Dfast=1.24±0.02 μm2 s−1). (Inset) The distribution of D* values of 7,472 tracked UvrB molecules after exposure to ultraviolet light (UV). (b) Distribution of D* values of 9,456 tracked UvrB molecules in ΔuvrA cells, fitted with a three species model (constrained at D* values obtained for UvrB in WT cells; 0.11, 0.41 and 1.24 μm2 s−1) established that 11%±3% of UvrB molecules were immobile, 24%±4% diffusing slowly and 65%±7% fast diffusing. (Inset) The distribution of D* values of 1,606 tracked UvrB molecules after exposure to UV. (c) Fraction of immobile UvrB molecules extracted by fitting a three species model to the distributions of D* values for WT cells and cells overexpressing unlabelled UvrA. Errors represent s.e.m. from fits to three experimental repeats. (d) Dwell time distributions for immobile UvrB±UV after correction for photobleaching. To measure the UvrB binding in the absence of DNA damage we used a strain overexpressing unlabelled UvrA, resulting in recruitment of UvrB to non-damaged DNA. Dwell times were measured with 1 s exposures followed by 4 s delay. Errors represent s.e.m. of three experimental repeats.
