Fig 2: Screening for MRE11 SUMOylation sites.a Schematic of the SUMOylation sites in MRE11. The predicted high-rank sites analyzed by JASSA, SUMOplot, and GPS-SUMO software, and the mass spectrometry sites from a previous study were listed. b K255, K384, K416, and K467 mutations impaired MRE11 SUMOylation. HEK293T cells were transfected with His-SUMO2 and SFB-WT MRE11 or the indicated single point mutants, and then MRE11 SUMOylation levels were analyzed by denaturing pull-down and immunoblotting. c Cells were pretreated with 10 µM ATRi (VE-821) and 10 µM ATMi (KU-55933) for 2 h and further with 1 µM CPT for 8 h, followed by the analysis of MRE11 SUMOylation. d SUMOylation of 4KR (containing K255R, K384R, K416R, and K467R) under CPT treatment or PIAS1 overexpression were analyzed. e Purified 4KR protein was analyzed by Coomassie blue staining, and then subjected to SUMOylation assay in vitro.

Fig 3: A working model of the MRE11 SUMOylation in DNA damage repair.The PIAS1-SENP3 axis dynamically controls the SUMOylation and ubiquitylation levels of MRE11 to facilitate DNA end resection. Upon DSB induction, the SUMO E3 ligase PIAS1 and MRE11 are recruited to broken DNA ends, and MRE11 SUMOylation is enhanced to antagonize ubiquitin-mediated proteasomal degradation during DNA end resection. Mainly after DNA end resection, SENP3 deSUMOylates MRE11 and prevents the excessive accumulation of MRE11.

Fig 4: The molecular mechanism of SUMO E3 ligase PIAS1 in HIRI through regulating NFATc1/HDAC1/IRF-1/p38 MAPK signaling pathway.

Fig 5: IAV PB2, PB1, and NP are differentially SUMOylated in HEK293T cells expressing exogenous SUMOylation components.(A-I) HEK293T cells were transfected with plasmids expressing HA-tagged WSNPB2 (A-C), WSNPB1 (D-F), or WSNNP (G-I), along with or without Flag-SUMO1/2/3, Ubc9-V5, and Myc-PIAS1 or its mutant. HEK293T cells transfected to express only Flag-SUMO1/2/3, Ubc9-V5, and Myc-PIAS1 served as a negative control. At 36 h post-transfection, cell lysates were immunoprecipitated with a mouse anti-HA mAb (A-C) or a rabbit anti-HA pAb (D-I), and were then subjected to western blotting with a rabbit anti-PB2 pAb (A-C), a mouse anti-PB1 mAb (D-F), a rabbit anti-NP pAb (G-I), and a mouse anti-Flag mAb (A-I) for the detection of PB2, PB1, NP, and SUMO1/2/3, respectively. Red triangle indicates the corresponding SUMOylated viral protein.