Fig. 3: Modulation of ASCC3^HR helicase, ATPase, and DNA binding activities by TRIP4.

a Stopped-flow/fluorescence-based DNA unwinding assays (no trap), showing that TRIP4, but not TRIP4^1–230 or TRIP4^403–581, stimulates ASCC3^HR DNA helicase activity. In this and analogous experiments in the following, curves show fits of the data to a double exponential equation (fraction unwound = A_fast*(1 – exp(–k_fast * t)) + A_slow * (1 – exp(–k_slow * t)); A_fast/slow, unwinding amplitudes of the fast/slow phases; k_fast/slow, unwinding rate constants of the fast/slow phases [s⁻¹]; t, time [s])³⁵. b Stopped-flow/fluorescence-based assays (no trap) monitoring DNA unwinding by ASCC3^HR constructs, in which either the NC (D611A) or the CC (D1463A) are inactivated, alone or in the presence of TRIP4, showing that both NC and CC exhibit helicase activities that are stimulated by TRIP4. Data for ASCC3^HR,D611A-based DNA unwinding had been reported previously¹⁰ and are reproduced here to facilitate direct comparison. c Apparent DNA-stimulated ATPase rates of ASCC3 constructs alone or in complex with TRIP4 (indicated at the bottom). HR, helicase region; NC, N-terminal cassette. Values represent means (bars) ±  SD (lines); individual data points (open circles) for n = 3 technical replicates are shown. Apparent ATPase rates were calculated as described in “Methods” and in Supplementary Fig. 7. Statistical significance was determined by unpaired, two-sided t tests. Significance indicators represent the significance of differences to wt ASCC3^HR; **P =  0.0049; ***P =  0.0001; ****P<  0.0001; ns not significant. ASCC3^HR constructs, in which either the NC (D611A) or the CC (D1463A) are inactivated, show reduced ATPase activities, and TRIP4 does not significantly enhance the ASCC3^HR ATPase. d Stopped-flow/fluorescence-based RNA unwinding assays (no trap), showing that ASCC3^HR can unwind RNA duplexes and that ASCC3^HR-based RNA unwinding is also modulated by TRIP4. e MST assays monitoring DNA binding by the indicated ASCC3 and TRIP4 variants or complexes. Data represent means ± SD; n = 3 (ASCC3^HR), n = 7 (ASCC3^HR-TRIP4), n = 6 (ASCC3^HR-TRIP4^1–230), n = 4 (TRIP4) or n = 6 (TRIP4^1–230) technical replicates. Curves show fits of the data to a Hill model (F_norm = F_norm,max * X^h / (EC50^h + X^h); F_norm,max, maximum F_norm value; X, concentration of the protein or protein complex; h, Hill slope; EC50, concentration needed to achieve a half-maximum binding at equilibrium). Source data are provided as a Source Data file.