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. 2015 Jan 6;6:5849. doi: 10.1038/ncomms6849

Figure 4. Rad4-induced DNA-opening dynamics measured by T-jump spectroscopy using 2AP fluorescence as a probe.

Figure 4

(a) 2AP (X in the schematic representation of DNA substrates) was placed within 3-bp mismatch DNA (AN3) and matched DNA (AN4; Supplementary Table 1). The 2AP fluorescence emission spectra were measured for DNA alone (black) and Rad4–DNA complexes (red) with excitation at 314 nm at 25 °C (left: AN3; right: AN4). The 2AP fluorescence emission intensities increase 4.0 (±0.7)-fold and 1.4 (±0.1)-fold on Rad4 binding to mismatch and matched DNA, respectively. All measurements were done with untethered Rad4. Protein and DNA concentrations were 10 μM each. (b) The maxima of the equilibrium 2AP fluorescence emission, measured at 365 nm, are plotted as a function of temperature for DNA alone (black) and Rad4–DNA complexes (red; left: AN3; right: AN4). Open and filled symbols are for two independent sets of measurements on each sample. In each panel, the intensities for free DNA and DNA in complex have been normalized to match at the lowest temperature. (c) Relaxation kinetic traces measured in response to a ~7 °C T-jump show (left) single-exponential kinetics, with relaxation time 5.1±0.5 ms (at final temperature 26 °C) for Rad4–mismatch DNA, and (right) much slower kinetics, with relaxation time 190±42 ms (at final temperature 29 °C) for Rad4–matched DNA. The uncertainties in the relaxation times are sample s.d. from two sets of measurements. Note that the Kd values of Rad4 bound to mismatch or matched DNA are in nanomolar range (Supplementary Fig. 1), well below the 60 μM concentrations used in the T-jump experiments. (d) Relaxation kinetics measured on the DNA-only samples: AN3 alone, in response to a 6 °C T-jump (left), and AN4 alone, in response to a 10 °C T-jump (right), exhibit much slower kinetics, with relaxation times 341±70 and 240±35 ms, respectively, consistent with the T-jump recovery kinetics (see also Supplementary Fig. 4d). The relaxation times for samples that exhibit only the slow kinetics were determined by making measurements over a longer time window, up to 80–320 ms.