SLX4 containing UBZ deletion fails to localize to sites of ICL induction in FA cells

We tested the ability of human SLX4 to localize along a track of ICLs induced by psoralen conjugates (Thazhathveetil et al., 2007). Human U2OS cells stably expressing GFP–SLX4 were exposed to trimethyl-psoralen (TMP) and micro-irradiated along a track in cell nuclei using a 355-nm UV-A laser. As shown in Fig. 1B, GFP–SLX4 (upper panels), but not GFP (middle panels), formed a stripe along the laser track, which also contained the phosphorylated histone variant γ-H2AX; no stripe was formed when cells had not been exposed to TMP (Fig. 1B, lower panels). Indirect immunofluorescence analysis with antibodies raised in-house (Wilson et al., 2013) revealed that endogenous SLX4 was also recruited to sites of psoralen–UVA laser (PUVA)-induced DNA damage (Fig. 1C). Every cell that had a stripe of γ-H2AX also displayed an SLX4 stripe (data not shown).

Next, we tested the role of the SLX4 UBZ domains in recruitment to sites of ICLs. Whereas SLX4 in normal human fibroblasts formed stripes along tracks of PUVA-induced DNA damage, SLX4 in fibroblasts from patients 457/1, 457/2 and 457/3 did not (Fig. 1D). By contrast, immunoblotting of SLX4 immunoprecipitates showed that SLX4 was expressed at close-to-normal levels in fibroblasts from FA patients 457/1–3, and that it interacted normally with XPF–ERCC1, MUS81–EME1, SLX1 and C20ORF94 (supplementary material Fig. S1A,B). These data suggest that at least one of the two SLX4 UBZ domains recognizes a ubiquitylated protein at DNA damage sites, leading to SLX4 recruitment.

Only one of the two SLX4 UBZ domains binds to ubiquitin polymers

We next wished to explore the relative contribution of the individual UBZ domains to SLX4 function, and we first investigated ubiquitin binding. Maltose-binding protein (MBP)-fused fragments of SLX4 containing both UBZ domains (UBZ-1+UBZ-2) were immobilized on amylose–agarose beads. Mutant proteins in which conserved cysteine residues in either the first or the second UBZ domain were mutated to alanine – C296A+C299A (UBZ-1*) and C336A+C339A (UBZ-2*), respectively – were used as controls (Fig. 2A). MBP-tagged SLX4 (UBZ-1+UBZ-2) showed robust binding to K63-linked poly-ubiquitin chains but not to K48-linked polymers (Fig. 2B). Mutating UBZ-1 abolished binding to K63-linked poly-ubiquitin, whereas mutating UBZ-2 had no effect. Similar data were obtained when the MBP tag was replaced with GST (supplementary material Fig. S2A,B). Consistent with these data, GST–UBZ-1 alone bound to K63-linked poly-ubiquitin, and mutating the conserved cysteine residues abolished binding, whereas GST–UBZ-2 alone did not bind to ubiquitin (supplementary material Fig. S2A,B). A minimum of six ubiquitin moieties was required for SLX4 (UBZ-1+UBZ-2) fused to MBP (Fig. 2C) or GST (supplementary material Fig. S2C) to bind to ubiquitin chains. Binding to mono-, di-, tri- or tetra-ubiquitin was much weaker. These data show that SLX4 domain UBZ-1, but not UBZ-2, binds to poly-ubiquitin chains with a preference for K63- over K48-linkages.

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Fig. 2.

Testing the ability of the SLX4 UBZ domains to bind to ubiquitin. (A) Diagram of the MBP-tagged SLX4 fragments used in ubiquitin-binding assays. Asterisks denote cysteine to alanine mutations at Cys 296 and Cys 299 in UBZ-1 (fragment 2) or Cys 336 and Cys 339 in UBZ-2 (fragment 3). (B) The fragments shown in A or MBP alone were immobilized on amylose–agarose and incubated with K48-linked or K63-linked poly-ubiquitin (pUb, 2–7) chains. Pulldowns were subjected to SDS-PAGE and immunoblotted (WB) with anti-ubiquitin antibodies. The bottom panel shows a Ponceau staining of the membrane performed prior to blotting. (C) Same as for B, except that mono-ubiquitin or K63-linked poly-ubiquitin chains of the indicated length were used with fragments 1–3.

Recruitment of SLX4 to sites of DNA damage requires the UBZ-1 domain, but not the UBZ-2 domain, FANCD2, RNF8, RAD18 or BRCA1

We next tested the effect of UBZ mutations on the localization of mouse SLX4 expressed in murine embryonic fibroblasts (MEFs) from Slx4^−/− mice (Castor et al., 2013). Slx4^−/− MEFs were infected with viruses containing wild-type SLX4 fused to GFP or SLX4 bearing mutations in UBZ-1 (UBZ-1*) or UBZ-2 (UBZ-2*). Both wild-type SLX4 and SLX4-UBZ-2* were recruited to the sites of PUVA-induced ICLs, but the SLX4-UBZ-1* mutant was not recruited to sites of DNA damage in any of the cells in the population that had been micro-irradiated (Fig. 3A; data not shown). Consistent with these data, UBZ-1 alone fused to both a nuclear localization signal and GFP was capable of localizing at sites of PUVA-induced DNA damage, similar to wild-type SLX4, although on average the intensity of the stripes was weaker than that of stripes observed in cells containing wild-type SLX4 (supplementary material Fig. S3).

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Fig. 3.

Relative contributions of the SLX4 UBZ domains to DNA repair and recruitment to DNA damage sites. (A) Slx4^−/− MEFs were infected with retroviruses expressing GFP-tagged mouse (m)SLX4 wild-type (WT), SLX4 UBZ-1* or SLX4 UBZ-2*. Cells were incubated with trimethyl-psoralen (TMP) and subjected to subnuclear micro-irradiation using a 355-nm UV-A laser. Cells were fixed and subjected to indirect immunofluorescence analysis with antibodies against GFP or γ-H2AX. (B) PD20 cells lacking FANCD2 (upper panels), or PD20 cells stably expressing FANCD2 (middle panels) or FANCD2 K561R (lower panels), were subjected to laser micro-irradiation as in A. Endogenous SLX4 and γ-H2AX were analyzed. (C) Same as for B, except that U2OS cells stably expressing RNF8 shRNA plus parental cells, HCT-116 cells lacking RAD18 plus parental cells or UWB1.289 cells lacking BRCA1 were used. UWB1.289 cells expressing BRCA1 were used as a control. For each population, ∼300 cells were counted, and representative images are shown. Scale bars: 10 µm.

Mono-ubiquitylation of FANCD2 was reported to mediate recruitment of chicken SLX4 to subnuclear foci in DT-40 cells (Yamamoto et al., 2011). These data are at odds, however, with the preference of the human SLX4 UBZ-1 domain for ubiquitin polymers over mono-ubiquitin (Fig. 2C). In this light, we found that SLX4 could form normal stripes along tracks of DNA damage induced by PUVA treatment in FANCD2^−/− (PD20) human cells transfected with empty vector, in a manner that was indistinguishable from PD20 cells expressing wild-type FANCD2 or a mutant K516R FANCD2 that could not be ubiquitylated (Fig. 3B) (Garcia-Higuera et al., 2001).

Besides the FA core complex that ubiquitylates FANCD2 and FANCI, several other E3 ligases have been implicated in ICL repair, including RAD18, which promotes FANCD2 ubiquitylation (Geng et al., 2010; Song et al., 2010), RNF8, which promotes histone ubiquitylation and recruitment of FAAP20 (Leung et al., 2012; Yan et al., 2012), and BRCA1, although the E3 ligase activity of this protein might not be required for DNA repair (Aressy and Greenberg, 2012). SLX4 recruitment to sites of PUVA-induced damage in (1) U2OS cells stably expressing an RNF8 shRNA that prevented 53BP1 recruitment (supplementary material Fig. S3), (2) HCT-116 cells in which RAD18 was disrupted and (3) UWB1.289 cells, which are null for BRCA1, was indistinguishable from that of the relevant parental control cells; every cell with a stripe of γ-H2AX also had an SLX4 stripe of normal intensity (Fig. 3C). Taken together, these data show that SLX4 is recruited to the sites of DNA damage through its UBZ-1 domain, but the ubiquitylated ligand in question is not FANCD2 in human cells and recruitment does not require the E3 ligases that have already been implicated in ICL repair.

SLX4 domain UBZ-1, but not UBZ-2, is required for ICL repair

We next tested the effect of UBZ domain mutations on the sensitivity of cells to mitomycin C (MMC), as a readout of ICL repair. Slx4^−/− cells infected with empty pBABE-puro vector were much more sensitive to MMC than were wild-type cells infected with the same virus (Fig. 4A). Furthermore, the MMC hypersensitivity of Slx4^−/− cells was rescued by untagged wild-type mouse SLX4. The SLX4 UBZ-1* mutant was barely capable of rescuing the MMC sensitivity of Slx4^−/− cells, but the UBZ-2 mutant behaved like wild-type SLX4 (Fig. 4A). Consistent with these data, Slx4^−/− MEFs showed an increased incidence of chromosome abnormalities (defined as breaks and radial chromosomes), which was rescued by wild-type SLX4. Mutating domain UBZ-1 completely prevented rescue, whereas mutating UBZ-2 made no difference (Fig. 4B). Taken together, these data show that SLX4 domain UBZ-1, but not UBZ-2, is vital for the repair of ICLs.

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Fig. 4.

Relative contributions of the SLX4 UBZ domains to the resolution of Holliday junctions. (A) Clonogenic survival analysis of Slx4^−/− MEFs stably expressing untagged mouse (m)SLX4, SLX4 UBZ-1* or SLX4 UBZ-2*, exposed to the indicated doses of MMC. For each genotype, the viability of untreated cells was defined as 100%. Wild-type MEFs and Slx4^−/− MEFs infected with empty virus (+vector) were used as controls. Data represent the mean±s.e.m., n = 3. (B) Metaphase spreads of MEFs treated with MMC (20 ng/ml) were stained with DAPI and analyzed for the presence of radial and broken chromosomes. Each cell line was either treated with MMC or left untreated, and 50 metaphase spreads were analyzed for each condition to determine the number of abnormalities per metaphase. WT, wild-type. Data show the mean±s.d. (C) Wild-type (Slx4^+/+) MEFs, Slx4^−/− MEFs and Slx4^−/− MEFs infected with retroviruses expressing wild-type SLX4 (WT) or SLX4 bearing mutations in domains UBZ-1 (UBZ-1*) or UBZ-2 (UBZ-2*) were exposed to MMC (10 ng/ml) and the frequency of SCEs was measured. (D) Same as for C, expect that SCE frequencies in MEFs were measured after the depletion of BLM. In C and D, 400 chromosomes in three metaphase spreads (1200 in total) were analyzed. Data represent the mean±s.e.m.

Both SLX4 UBZ domains are required for the resolution of Holliday junctions

SLX4 is required for around one third of the sister chromatid exchanges (SCEs) induced by MMC in MEFs (Castor et al., 2013), and we next tested the effect of UBZ domain mutations on the occurrence of SCEs. As shown in Fig. 4C, MMC raises the frequency of SCEs in wild-type MEFs by around 2.5 fold. Compared with wild-type MEFs, Slx4^−/− MEFs showed ∼30% fewer SCEs after treatment with MMC, but this defect was rescued by wild-type SLX4 or SLX4-UBZ2*. No such rescue was observed with the version of SLX4 bearing mutations in UBZ-1 (Fig. 4C).

SLX4 is required for the nucleolytic resolution of Holliday junctions that escape dissolution by the BTR complex (Castor et al., 2013; Wyatt et al., 2013). We next examined the requirement of the SLX4 UBZ domains for resolution of Holliday junctions in this context. Short hairpin (sh)RNA-expressing retroviruses were used to deplete BLM from Slx4^−/− MEFs (Castor et al., 2013) (supplementary material Fig. S4). Depletion of BLM from wild-type MEFs caused an increase of ∼2.5 fold in SCE frequency but this increase was severely blunted in Slx4^−/− MEFs (Fig. 4D). Wild-type SLX4 rescued this defect, but mutating either the first or the second UBZ domain completely prevented rescue (Fig. 4D). These data suggest that both of the UBZ domains in SLX4 are required for the resolution of Holliday junctions that escape dissolution.

In this study, we showed that the mutated form of SLX4 expressed in the cells of German siblings 457/1–3, which lacks all of the second UBZ domain and part of the first, is not recruited to sites of DNA damage. By analyzing the two domains individually, we found that UBZ-1, but not UBZ-2, binds to ubiquitin polymers, mediates recruitment of SLX4 to sites of PUVA-induced DNA damage and is essential for both efficient ICL repair and SCEs induced by ICLs in MEFs. Therefore, proper targeting of SLX4, mediated by UBZ-1, appears to be essential for ICL repair, and it is likely that failure to correctly target SLX4 explains the FA phenotype in patients 457/1-3.

What is the ubiquitylated ligand at sites of DNA damage that recruits SLX4 through UBZ-1? The mono-ubiquitylated form of FANCD2 was reported to be required for the formation of foci in DT-40 chicken cells exposed to MMC (Yamamoto et al., 2011). In the present study, we found that the recruitment of SLX4 to sites of DNA damage induced in this manner occurs normally in cells that lack FANCD2 and in cells lacking RAD18, which is required for FANCD2 ubiquitylation (Geng et al., 2010; Song et al., 2010). These data are consistent with our observation that SLX4 only binds to poly-ubiquitin chains of at least six ubiquitin moieties, whereas FANCD2 is mono-ubiquitylated, not poly-ubiquitylated. We predict that there exists a poly-ubiquitylated ligand that recruits SLX4 to sites of ICLs. It will be interesting to identify this protein as well as the relevant E3 ligase involved, which appears to be distinct from any of the E3 ligases previously implicated in ICL repair.

Although SLX4 UBZ-2 is dispensable for ICL repair, it is required, together with UBZ-1, for the resolution of Holliday junctions that escape dissolution by the BTR complex. Recent data from our lab suggests that the role of the SLX4 complex in repairing ICLs is independent of Holliday junction cleavage and probably reflects the cleavage of alternative branched DNA repair intermediates (Castor et al., 2013). The observation in the present study that SLX4 UBZ-2 is required for the resolution of Holliday junctions that escape dissolution by BTR, but not for ICL repair, is consistent with this idea. Even though UBZ-2 appears to have all of the residues necessary for ubiquitin binding, it does not bind to mono-ubiquitin or to K48- or K63-linked poly-ubiquitin chains in vitro (Fig. 2). It is possible, however, that this domain binds to atypical poly-ubiquitin chains such as K11, K27 or K33 linkages or to a ubiquitin-like domain in the protein that recruits SLX4 to sites of DNA damage. Finding the ligands for both of the SLX4 UBZ domains will be an interesting area of investigation.