FIGURE 5.
RAD50 ATP hydrolysis prevents shutdown of the MRE11 exonuclease activity. A, shown is a schematic representation of the DNA hairpin substrate used in the assays. B, the exonuclease reactions comprise 2 μm MRE111–342 or L4-MRE11 protein, 200 μm nucleotide, and the indicated concentrations of 2-AP DNA substrate. Samples were incubated at 55 °C. After 2, 4, 6, 10, 15, and 20 min, 20-μl aliquots were removed and quenched, and the released 2-AP was measured in TECAN plate reader using excitation and emission wavelength of 310 and 375 nm, respectively. The intensities used to determine the turnover number/min/protein monomer were always measured in the linear range of a calibration curve from the fluorescence intensity of free 2-amino purine. Data were fitted (solid lines) using Michaelis-Menten equation. The added ions are indicated (B–E).