Fig 2: Scheme depicting role of HORMAD1 in DSB signaling and repair. Our immunofluorescence microscopy analyses (Fig. 4) show that formation of CtIP, RAD51 and RPA p34-containing IRIF is reduced in HORMAD1-depleted cancer cells (whereas 53BP1 and p95/NBS1 foci remain intact in the absence of HORMAD1). We infer that HORMAD1 contributes to HR pathway activation at (or prior to) the CtIP-mediated resection step, but does not affect the proximal stages of DSB signaling. HORMAD1-depletion leads to attenuation of DR-GFP/HR reporter activity (Fig. 3), prolonged IR-induced ATM signaling (Fig. 4), and radiosensitivity (Fig. 3), further demonstrating that HORMAD1 promotes the HR pathway.

Fig 3: PCNA mono-ubiquitination during G1 is RPA-dependent and p95-independent. (A) Replicate cultures of HDF were transfected with siRNA targeting p95 or with a scrambled control siRNA. The control and p95-depleted cells were synchronized in G1 and S-phase and treated with genotoxins. Chromatin extracts were isolated 1 h after genotoxin treatments and analysed by SDS–PAGE and immunoblotting with the indicated antibodies. (B) Replicate cultures of HDF were transfected with siRNA targeting RPA34, RNF8 or with a scrambled control siRNA. The resulting cells were synchronized in G1 and S-phase and treated with genotoxins. Chromatin extracts were isolated and analysed by SDS–PAGE and immunoblotting with the indicated antibodies. Bands corresponding to chromatin-bound RPA34 were quantified by densitometry. The amount of RPA34 in each lane is expressed relative to the amount of RPA34 in scrambled control siRNA-treated HDF in G1 that did not receive DNA damage.

Fig 4: MRE11 is UFMylated and this UFMylation transiently increases after DSB induction. (A) MRE11 is UFMylated in vivo. HEK293T cells co-expressing UFMylation factors (UBA5, UFC1, UFL1, UfBP1, HA-UFM1?C2) and GFP-MRE11 were lysed in 5% SDS-containing buffer. Total cell lysates were boiled for 5 min before IP and immunoblotting with the indicated antibodies. (B and C) MRE11 UFMylation is induced by DSBs. Experiments were performed as described in A except that HEK293T transfectants were treated with 10 µg/ml bleomycin (B) or 5-Gy IR (C) and total cell lysates were harvested at different time-points post-treatment. (D) UFMylated MRE11 is enriched on chromatin. Chromatin fractionation assays were performed with HEK293T cells co-expressing the UFMylation factors (UBA5, UFC1, UFL1, UfBP1, HA-UFM1?C2) and GFP-MRE11. Both the cytosolic fraction (SF) and chromatin-enriched fraction (CF) were subjected to IP with an anti-GFP antibody and immunoblotting with the indicated antibodies. (E) UFMylation of NBS1 was undetected. HEK293T cells co-expressing the UFMylation factors (UBA5, UFC1, UFL1, UfBP1, HA-UFM1?C2) and FLAG-MRE11 or FLAG-NBS1 were lysed in 5% SDS-containing buffer. Total cell lysates were boiled for 5 min before IP and immunoblotting with the indicated antibodies. (F) MRE11 directly interacted with UFL1 in vitro. Bacterially produced GST or GST-MRE11 was used to pull down bacterially produced HIS-UFL1. (G) MRE11 is UFMylated in vitro. Recombinant UFMylation factors (HIS-UBA5, HIS-UFC1, HIS-UFL1 and HIS-HA-UFM1?C2) with bacterially produced GST, GST-MRE11 or GST-MRE11(K282R) were incubated in UFMylation buffer at 30°C for 90 min. The reaction was terminated by adding SDS sample buffer and the samples were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting with the indicated antibodies.

Fig 5: UFMylation promotes MRN complex formation under unperturbed conditions. (A) UFL1 depletion decreased MRN complex formation. Total lysates derived from HEK293T cells transfected with UFL1 (siUFL1 1# and 2#) or control (siCTR) siRNAs were immunoprecipitated with RAD50 or NBS1 antibodies. The precipitates were probed with the indicated antibodies. (B) Over-expression of UfSP2 compromised MRN complex formation. Total lysates derived from HEK293T cells overexpressing HA-VEC, HA-UfSP2 or HA-UfSP2(C302S) were subjected to IP and immunoblotting with the indicated antibodies. Abbreviations: bleo, bleomycin; IB, immunoblot; IP, immunoprecipitation; siCTR, small interfering RNA control.