Fig. 5: MRE11 SUMOylation is essential for efficient HR and cell viability.

a, b HeLa cells with SUMOylation-deficient MRE11 exhibited impaired pRPA2 (S4/S8) and RAD51 foci formation. Scale bar, 10 μm. The data are presented as means ± SD, n (Vector+siCon; Vector+siMRE11; WT + siMRE11; 4KR + siMRE11) = 131; 131; 125; 135 cells for a; n (Vector+siCon; Vector+siMRE11; WT + siMRE11; 4KR + siMRE11) = 112; 112; 108; 108 cells for b, ns = no significance. c shMRE11 HeLa cells expressing ectopic SFB-WT and the indicated MRE11 mutants were treated with 1 μM CPT for 1 h or not. Then, cell lysates were subjected to immunoblotting. d DR-GFP U2OS cells stably co-expressing SFB-Vector/SFB-WT/SFB-4KR and shMRE11 were infected with I-SceI lentivirus for 48 h, followed by flow cytometric analysis for HR efficiency (means ± SEM, n = 3 independent experiments). e–g The cell viability of stable SFB-WT and SFB-4KR cells with endogenous MRE11 knocked down was detected by CCK-8 assay and colony formation assay (means ± SEM, n = 3 independent experiments, ns = no significance). For CCK-8 assay, cells were treated with the indicated concentrations of CPT/cisplatin for 36 h or 1 μM CPT/cisplatin for the indicated times. h The assembly of MRN complex containing 4KR MRE11 was assayed by Flag pull-down in vitro. The supernatant (S) containing unbound protein and the eluate (E) were subjected to immunoblotting. i The interactions among 4KR MRE11, RAD50 and NBS1 in HEK293T cells were analyzed by co-IP with S-beads. j The indicated concentrations of WT and 4KR MRE11 were incubated with DNA for 30 min, and the nuclease products were resolved in denaturing polyacrylamide gels. Quantification of the results is presented (means ± SD, n = 3 independent experiments, ns = no significance). k Time-course analysis of DNA resection by WT and 4KR MRE11 (10 nM). Electrophoresis and quantification were performed as in j.