Extended Data Fig. 2: RAD51 interacts directly to HELQ and promotes its helicase activity.

a, SDS-PAGE gel (4-12%) showing purified recombinant human RAD51 from E. coli. The gel was stained with CBB. We used two preparations of RAD51 in this study. b, Protein interaction analysis of MBP-HELQ-Flag and RAD51 using both amylose and Flag pull-down assay. The final eluates were run on SDS-PAGE gel (4-12%) and stained with CBB. The interaction analysis was repeated 3 times with similar results. c-e, Representative native gels of DNA unwinding of D-loop, Y-structure and lagging strand fork substrates with HELQ (1 nM) and indicated concentrations of either RAD51 or RecA. f, Representative native gel of DNA unwinding of 3′-overhang with indicated concentrations of RecA and HELQ (1 nM). g, Quantification of experiments such as shown in f. n = 4 independent experiments; mean ± S.E.M. h, SDS-PAGE gel (4-12%) showing purified recombinant GST and GST-BRC4 peptides from E. coli. The gel was stained with CBB. Single preparation of GST and GST-BRC4 used in this study. i-j, EMSA gels (6% polyacrylamide) showing RAD51 binding to 3′-overhang and Y-structure in the presence and absence of indicated concentration of GST-BRC4 and GST peptides. k, Representative native gel of DNA unwinding of 3′-overhang by HELQ with RAD51 and indicated concentrations of GST-BRC4 and GST peptides. l, Quantification of experiments such as shown in k. n = 4 independent experiments; mean ± S.E.M. m, Representative native gel of DNA unwinding of Y-structure by HELQ with RAD51 and indicated concentrations of GST-BRC4 and GST peptides. n, Quantification of experiments such as shown in m. n = 4 independent experiments; mean ± S.E.M. o, Representative native gel of DNA unwinding of Y-structure by HELQ with inhibitory concentration of RAD51 and indicated concentrations of for substrates GST-BRC4 and GST peptides. p, Quantification of experiments such as shown in o. n = 3 independent experiments; mean ± S.E.M. q, Schematics representation of quenching-based kinetic DNA unwinding assay of 3′-overhang. Initially, oligo F (49-mer), labelled at 5′ end with fluorescein (F), is annealed with oligo R, which is labelled with rhodamine (R) at 3′ end. Due to close proximity, FRET from fluorescein is quenched by rhodamine constitutively resulting in low FRET signal. Upon DNA unwinding, DNA strands are separated and thus rhodamine no longer able to quench fluorescein, results in higher FRET signal. r, Relative unwinding of 3′-overhang with HELQ and indicated concentrations of RAD51 as determined by quenching-based kinetic assay. n = 3 independent experiments; shaded area represents mean ± S.E.M.; black lines represent exponential or linear fits. s, Relative unwinding of 3′-overhang with HELQ and indicated concentrations of RecA as determined by quenching-based kinetic assay. n = 3 independent experiments; shaded area represents mean ± S.E.M.; black lines represent exponential or linear fits.