Fig 1: WASp directly binds RPA and enables RPA binding to ssDNA.a, ELISA. In vitro protein-protein interaction monitored by ELISA for the indicated purified proteins at the indicated concentrations. 1st protein is coated onto the plate at a fixed concentration; 2nd protein is added at the indicated increasing concentrations. hRPA1-3, human heterotrimeric complex of RPA1, 2, 3; hMAX, human Myc-associated factor X; BSA, bovine serum albumin; ScRpa, Saccharomyces Rpa; WT*WASp, wild-type WASp. The displayed data are mean+SEM, n = 5 independent assays. The intrinsic property of WASp molecules to spontaneously oligomerize (via VCA:VCA-domain interaction) served as +ve control, and is shown for the highest concentration (1 µg) of WT*WASp protein. b Schematic of the multi-modular domain structure of human WASp showing WH1-domain, Basic-domain containing the NLS (B), GTPase-binding domain (GBD), Polyproline-domain (Pro), VCA-domain (WH2, C, A subdomains), in which the location of evolutionarily conserved RPA1-binding motif (RBM1) within the VCA-domain is shown. WH2 (aka, V region) binds monomeric G-actin, C and A regions bind Arp2/3-complex. Amino-acid alignments of human WASp’s RBM1 with those of other proteins and species are shown, in which residues that are conserved (in evolution) and common (with other RPA1-binding proteins) are highlighted in blue. The prototypical RBM1-consensus is shown at the top, D: Aspartic acid; ?: hydrophobic or charged side-chain amino acid; x: any amino acid. c ELISA. Shown is the binding efficiency of purified heterotrimeric RPA1-3 protein (on left) or purified Arp proteins (on right) with either purified Wt*WASp or RBM1-deleted WASp mutant (?RBM1*WASp) at the indicated concentrations. Physical interactions of WT*WASp:WT*WASp and mutant ?RBM1*WASp:?RBM1*WASp (from spontaneous oligomerization of their respective VCA-domains) served as +ve controls. n = 3 independent assays, mean+SEM. p-value: ****<0.0001. ns, nonsignificant by Mann–Whitney unpaired two-tailed nonparametric. d Proximity ligation assay. Z-stack collapsed composite confocal IF images showing PLA signals (dots) between the indicated protein pairs after transfecting WASp-deficient human T cells with GFP-tagged Wt*WASp or ?RBM1*WASp mutant after CPT-induced damage. The images/data are for the GFP+ cells enriched by FACS-sorting. Box plots are from n = 150 cells, Mann-Whitney unpaired two-tailed nonparametric p-value: ****<0.0001; * = 0.01; ns, nonsignificant. DAPI (in blue) demarcates the nucleus. Scale bar: 4 µm. EMSA. Assays performed using the indicated purified proteins at the indicated concentrations (nM) against fixed concentration of ssDNA (panel e) or other 3’-ended DNA structures (panel f). Data is representative of at least 3 replicates per condition. Red arrows indicate antibody super-shifted RPA band; other arrows indicate location of non-shifted RPA bands. Other related EMSA results are shown in Supplementary Fig. S2.