Fig 2: Characterization of the KO30 cell line expressing an N-terminally truncated form of SLX4. (A) Western blot showing that HeLa FITo KO1 and KO30 clones generated by CRISPR-Cas9 express a truncated form of SLX4 termed SLX4ΔNter (indicated by an arrow), the expression of which is sensitive to a siRNA that targets SLX4 mRNA. (B) SLX4ΔNter protein starts at Methionine 360; see supplementary results for details. (C) Clonogenic survival assay in response to mitomycin C (MMC) of HeLa FITo, KO30 cells and KO30 cells complemented with FHA-SLX4 WT or UBZ-mutated (UBZmut). Cells were treated for 24 h with the indicated dose of MMC (n = 2–4 experiments, mean ± SD are represented).

Fig 3: Abraxas limits K63-linked ubiquitin-dependent SLX4/MUS81 recruitment to CPT damage sites.a Increased chromatin-bound MUS81 in Abraxas KO cells was reduced by SLX4 knockdown. Cells transfected with indicated siRNAs were treated with 1 μM CPT for 1 h. Chromatin fraction was isolated for analysis by western blottings. b SLX4 knockdown reduced the elevated RPA32-pS4/8 levels in Abraxas KO treated with CPT. Total lysates were used for western blottings. c K63-linked polyubiquitination is increased in Abraxas KO U2OS cells treated with CPT. WT and Abraxas KO U2OS cells were untreated or treated with 1 μM CPT for 1 h. Total cell lysates and chromatin fractions were prepared from samples for western blottings. K63-linked ubiquitination was detected by antibodies against K63-linked chain. d BRCC36 knockdown leads to increased RPA32-pS4/8 and overloading of SLX4/MUS81 to CPT-damaged chromatin. Total cell lysates and chromatin fractions were prepared from cells transfected with indicated siRNAs and treated with CPT (1 μM, 1 h). e Ubc13 knockdown reduced elevated RPA32-pS4/8 levels and chromatin-bound MUS81 in Abraxas KO cells treated with CPT. f RNF168 knockdown reduced elevated RPA32-pS4/8 levels and chromatin-bound MUS81 in Abraxas KO cells treated with CPT. See also Supplementary Fig. 5.

Fig 4: Interaction of SLX4 and MSH2 contributes to the toxicity of 6-thioguanine (6-TG) or N-methyl-N’-nitro-N-nitrosoguanidine (MNNG). (A) Clonogenic survival assay of HeLa FITo cells transfected with the indicated siRNA in response to a 24 h treatment with 6-TG (n = 4–7 experiments, mean ± SEM are represented). (B) Clonogenic survival assay in response to 6-TG of HeLa FITo, KO30 cells and KO30 cells complemented with FHA-SLX4 WT or SLX4ΔMSH2bd (n = 3–5 experiments, mean ± SEM are represented). (C) Clonogenic survival assay in response to MNNG of KO30 cells complemented with FHA-SLX4 WT or SLX4ΔMSH2bd (n = 3–4 experiments, mean ± SEM are represented). (D) KO30 cells complemented with FHA-SLX4 WT or SLX4ΔMSH2bd were treated with MNNG (0.1 μM) for 24 h before drug removal and addition of fresh medium. Cells were collected at the indicated time points of recovery (+rec) and induction of the DNA damage response was analysed by western blot.

Fig 5: A proposed model for the role of Abraxas/BRCA1-A complex.Abraxas/BRCA1-A complex inhibits DNA end resection and limits BIR by restricting SLX4/MUS81 chromatin loading through counteracting K63-linked ubiquitin modification catalyzed by RNF168/Ubc13 in response to CPT. In the absence of Abraxas, increased K63-linked ubiquitin conjugation leads to SLX4/MUS81 overloading on chromatin, generating increased seDSBs, which are further processed by MRE11 endonucleases, CTIP and DNA2/BLM, forming an extensive length of ssDNA. Uncontrolled MUS81 loading and excessive end resection also involve RAD52- and POLD3-dependent BIR that results in increased mitotic DNA synthesis, extensive chromosome aberrations, and cell lethality in Abraxas-deficient cell.