Fig. 4. MRNIP condensates incorporate the MRN complex and relocalize to damaged DNA.

a MRNIP-GFP droplets compartmentalized DNA from solutions. MRNIP-GFP droplets that formed in vitro were incubated with DNA solutions and fractionated by centrifugation. b γ-H2A.X localized within MRNIP droplets in cells after 15 min recovery post irradiation. MRNIP-GFP-expressing HeLa cells were irradiated (2 Gy) and subjected to γ-H2A.X detection at indicated time. c The quantification of γ-H2A.X foci area in (b). Colocalized, γ-H2A.X foci colocalized with MRNIP puncta; Surrounding, γ-H2A.X foci didn’t colocalize with MRNIP puncta. Data are presented as means ± SEM. Colocalized, n = 30; Surrounding, n = 61. Two-tailed unpaired Student’s t test. d MRNIP-GFP condensates moved to the microirradiation-induced DNA damage site. IR, microirradiation; mock, no IR. HeLa cells were transfected with plasmid and incubated with 10 μM BrdU for 24 h before microirradiation. e MRNIP-GFP droplets compartmentalized the MRN complex from the nuclear extract of HeLa cells. The indicated protein was incubated with 10 µg of HeLa nuclear extract in buffer containing 150 mM NaCl (pH 7.4) for 20 min at 20 °C and fractionated by centrifugation. f MRNIP droplets incorporated with MRE11 in cells. Live-cell image of cells co-expressing MRE11-mCherry and MRNIP-GFP. HeLa cells were transfected with indicated plasmids for 24 h before observation. g Co-immunoprecipitation assay showed that IDR1-deleted MRNIP did not interact with MRN complex. h MRNIP-GFP droplets incorporated MRE11 and dsDNA in vitro. The indicated proteins or dsDNA were incubated in buffer containing 150 mM NaCl, pH 7.4, for 20 min at 20 °C. Rho Rhodamine.