FIGURE 4.

DNA binding and DNA-activated ATPase activity of wild-type and mutant RecQ constructs. A, stopped-flow Trp fluorescence traces of the interaction of RecQ constructs (wild-type RecQ (black), RecQ^Y555A (dark gray), RecQ⁵²³ (gray), and RecQ⁴¹⁴ (light gray); all proteins applied at 0. 5 μm) with dT₅₄ (81 μm, nt) at 5 °C. Fluorescence levels were normalized to that of the DNA-free state. Lines show triple (wild-type RecQ) or double exponential (all mutants) fits to the data sets. (The slowest phase arose from photobleaching in each case; see “Results.”) B and C, comparison of fits comprising one (B, gray line) and two (B, black line) exponential DNA-binding phases (in addition to the slow photobleaching phase) to the wild-type RecQ trace (cf. A). Residuals of the fits shown in the main panel of C (gray and black, respectively) indicated systematic deviation from data points when fitting a single binding phase (gray). The inset of C shows that, in the case of RecQ⁵²³, the residuals of fits comprising either one (gray) or two (black) binding phases showed no systematic deviation from data points (fitted lines were indistinguishable; cf. A). This finding applied to all mutant constructs. D and E, dT₅₄ concentration dependence of rapid phase rate constants (k_rapid; D) and total amplitudes (A_total; E) of transients recorded at 5 °C as in A (wild-type RecQ (black squares), RecQ^Y555A (dark gray circles), RecQ⁵²³ (gray triangles), and RecQ⁴¹⁴ (light gray inverted triangles)) alongside linear (D) and hyperbolic (E) fits. (RecQ^Y555A and RecQ⁴¹⁴ amplitude data could not be reasonably fitted due to noise in the low values.) F, dT₅₄-activated ATPase activity of RecQ constructs (wild-type RecQ 15 nm, mutant constructs 10 nm; symbols as in D) as determined by a PK-LDH assay at 25 °C. Quadratic (wild-type RecQ, RecQ^Y555A) and hyperbolic (RecQ⁵²³ and RecQ⁴¹⁴) fits are shown as lines. Parameters derived from fits are shown in Table 3.