Fig 2: K282 is the essential UFMylation site in MRE11. (A) Mapping the essential regions for MRE11 UFMylation. A series of deletion mutants in the context of full-length MRE11 were generated as indicated. The mutants or the control plasmids were co-transfected with the UFMylation factors (UBA5, UFC1, UFL1, UfBP1, HA-UFM1ΔC2) into HEK293T cells. Experiments were performed as described in 5A but using these deletion mutants. (B) K282 is the major UFMylation site for MRE11. HEK293T cells co-expressing the UFMylation factors (UBA5, UFC1, UFL1, UfBP1, HA-UFM1ΔC2) and GFP-MRE11 or GFP-MRE11(K282R) were lysed in 5% SDS-containing buffer. Total cell lysates were boiled for 5 min before IP and immunoblotting with the indicated antibodies. (C) The pathogenic mutant MRE11(G285C) exhibited defective UFMylation. Experiments were performed as described in B except that GFP-MRE11(G285C) was also included.

Fig 3: UFMylation promotes MRE11 recruitment to the DNA damage stripes. (A and B) UFL1 depletion decreased the initial recruitment of MRE11 to DNA damage stripes. UFL1-depleted (siUFL1) or mock (siCTR)-depleted DU145 cells transiently expressed GFP-MRE11. (C and D) UfSP2 over-expression decreased MRE11 initial recruitment to DNA damage stripes. DU145 cells were co-transfected with an HA vector, HA-UfSP2 or HA-UfSP2(C302S) and GFP-MRE11 at a molar ratio of 10:1. GFP-positive cells were micro-irradiated with a UV laser (365 nm) and consecutive images were collected at 10-s interval for 10 min. Representative images of GFP-MRE11 recruitment are shown in A and C, and the statistical analysis of recruitment dynamics with the Spearman’s rank-order correlation test is shown in B and D.

Fig 4: MRE11 UFMylation promotes HR-medicated DSB repair and genome stability. (A) Depletion of UFL1-sensitized cells to bleomycin treatment. A549 cells stably expressing shCTR, shUFL1 or shUFL1 reconstituted with an shUFL1-resistant form of FLAG-UFL1 were treated with increasing concentrations of bleomycin, as indicated. The surviving colonies were analyzed after 10 days. (B and C) Depletion of UFL1 resulted in aberrant mitotic chromosomes. The cells generated in A were used for mitotic spread preparation, and >400 mitotic chromosomes per type of manipulated cells were analyzed. Representative images of mitotic spreads are shown in B, while the quantitative analysis is shown in (C). (D) Overexpression of UfSP2 sensitized cells to bleomycin treatment. A549 cells stably expressing a FLAG vector, FLAG-UfSP2 or FLAG-UfSP2(C302S) were treated with increasing concentrations of bleomycin, as indicated. The surviving colonies were analyzed after 10 days. (E) MRE11(K282R) failed to rescue MRE11-depletion-induced cellular sensitivity to bleomycin treatment. A549 cells stably expressing shCTR, shMRE11, shMRE11 reconstituted with FLAG-MRE11 or FLAG-MRE11(K282R) were treated with increasing concentrations of bleomycin, as indicated. The surviving colonies were analyzed after 10 days. (F) Defective MRE11 UFMylation compromised HR-mediated DSB repair. DRU2OS cells, in which a DR-GFP reporter cassette is integrated, stably expressing shCTR, shMRE11 or shMRE11 reconstituted with FLAG-MRE11, FLAG-MRE11(K282R) or FLAG-MRE11(G285C). HR assays were performed in triplicate. (G) Defective MRE11 UFMylation promoted NHEJ-mediated DSB repair. U2OS cells, in which an EJ5-GFP reporter cassette is integrated, stably expressing shCTR, shMRE11 or shMRE11 reconstituted with FLAG-MRE11, FLAG-MRE11(K282R) or FLAG-MRE11(G285C). NHEJ assays were performed in triplicate. (H and I) Defective MRE11 UFMylation contributed to aberrant mitotic chromosomes. A549 cells stably expressing shCTR, shMRE11 or shMRE11 reconstituted with FLAG-MRE11, FLAG-MRE11(K282R) or FLAG-MRE11(G285C) and then subjected to mitotic spread preparation; >400 mitotic chromosomes per type of manipulated cells were analyzed. All data were derived from three independent experiments. Representative images of mitotic spreads are shown in H, while the quantitative analysis is shown in I. A two-way ANOVA was performed to determine statistical significance. *: p<0.05; **: p<0.01; ***: p<0.001.

Fig 5: Defective UFMylation compromises optimal ATM activation upon DNA damage. (A and B) Inhibition of UFL1 expression compromised optimal activation of ATM upon DNA damage. A549 cells that were mock depleted (shCTR), UFL1 depleted (shUFL1) or UFL1 depleted, and then rescued with an shUFL1-resistant form of UFL1 (shUFL1-res) were treated with 10 μg/ml bleomycin (A) or irradiated with 5-Gy IR (B). Total cell lysates were harvested at different times after treatment and subjected to immunoblotting with the indicated antibodies. (C) Over-expression of UfSP2 compromised optimal activation of ATM upon DNA damage. A549 cells expressing FLAG-VEC, FLAG-UfSP2 or FLAG-UfSP2(C302S) were treated with 10 μg/ml bleomycin. Total cell lysates were harvested and subjected to immunoblotting with the indicated antibodies.