Fig. 1: Identification of MCM8IP by proximity-dependent biotin-identification (BioID) technology.

a Schematic of the protocol used to identify RPA1 interactors by BioID. HEK293T T-REx cells expressing doxycycline-inducible BirA*- or BirA*-RPA1 were treated with HU in the presence of exogenous biotin. Biotinylated proteins were captured in denaturing conditions from cell lysates by streptavidin pulldown and subjected to mass spectrometry for protein identification. b List of selected proteins identified by mass spectrometry that were enriched in streptavidin pulldowns conducted from BirA*-RPA1-expressing HEK293T T-REx cells relative to pulldowns performed from control BirA*-expressing cells. See also Supplementary Data 1. c Detection by western blot of MCM8IP in streptavidin pulldowns from HEK293T T-REx cells expressing BirA* or BirA*-RPA1. Cells were treated with HU (1 mM), cisplatin (20 µM), or olaparib (20 µM) in the presence of exogenous biotin for 24 h prior to lysis. Vinculin is shown as a loading control. d Detection by western blot of RPA1 and RPA2 following immunoprecipitation of HA-GFP or HA-MCM8IP from HEK293T cells. e Detection by western blot of RPA1 and MCM8IP (short exposure, s.e.; long exposure, l.e.) following subcellular fractionation of HCT116 cell lysates upon treatment with HU (1 mM), cisplatin (10 µM), or olaparib (10 µM) for 24 h. Tubulin and histone H3 are shown as loading and fractionation controls. f Representative images of FLAG-MCM8IP recruitment to sites of UV laser microirradiation in U2OS cells. DNA damage tracts are indicated with γH2AX staining. Scale bar = 20 µm. g Graphical representation of the percentage of FLAG-MCM8IP co-localizing with γH2AX following UV laser microirradiation in U2OS cells transfected with control or CtIP siRNA. The mean values ± SD of three independent experiments are presented. Statistical analysis relative to control siRNA was conducted using Student’s t-test (***p < 0.001, two-tailed).