SMARCAL1: a DNA repair protein recruited to double-strand breaks at stalled replication forks to promote genome stability through a homologous recombination pathway

doi:10.21203/rs.3.rs-5281240/v1

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SMARCAL1: a DNA repair protein recruited to double-strand breaks at stalled replication forks to promote genome stability through a homologous recombination pathway

https://doi.org/10.21203/rs.3.rs-5281240/v1

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To create an exact copy of the entire genome, replication should be completed promptly and accurately. However, obstacles can arise during replication that can cause genotoxic fork stalling. The DNA damage response pathway plays a crucial role in preventing fork collapse by stabilizing the stalled fork, regulating DNA repair, and promoting replication restart. There are multiple pathways to recover stalled replication forks, including double-strand break (DSB)-mediated recovery. SMARCAL1, a damage-binding protein involved in stabilizing stalled replication forks during DNA repair, is recruited to replication forks via interaction with replication protein A (RPA), the major ssDNA-binding protein in eukaryotic cells; however, the process by which it promotes DSB repair is poorly understood. Here, we present cellular and biochemical evidence that SMARCAL1 c.1920_1921insG frameshift mutation impairs SMARCAL1 protein expression causing defects in DNA damage response and hypersensitivity to several genotoxic agents that cause DSBs and replication fork collapse in a patient with Schimke immuno-osseous dysplasia. We analyzed the role of SMARCAL1 in the two major DNA DSB repair pathways, homologous recombination (HR) and nonhomologous end-joining (NHEJ). γ-H2AX foci repair kinetics analysis and survival experiments performed in SMARCAL1-deficient fibroblasts point to an essential role for SMARCAL1 in repairing DSBs generated in the S/G2 phase cell cycle as a result of oxidative damage and replication stress. The contribution to survival of SMARCAL1-dependent DSB rejoining in the S/G2 phase cell cycle suggests that SMARCAL1 is a novel damage repair protein involved in the HR pathway.

Radiosensitivity

DNA damage

SIOD

Immunodeficiency SIOD: Schimke immuno-osseous dysplasia

During DNA replication, cells employ multiple DNA repair and signaling mechanisms to ensure complete and accurate duplication of their genomes. Disruption of DNA replication activates a DNA damage response that slows cell cycle progression and promotes DNA repair to prevent genomic instability, one of the main factors responsible for cancer development. Replication stress response is a subset of the DNA damage response that operates during every S-phase when replication forks stall owing to DNA lesions keeping away from mutations or chromosome aberrations and protecting genome integrity (1–6).

The SNF2 ATPase family member SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A-like 1), also known as HARP (HepA-related protein) is a replication stress response enzyme involved in repairing damaged replication forks and restarting stalled forks (7–13). Replication fork repair is a complex process that can proceed through multiple pathways depending on the cause, persistence and genomic context of the replication stress. These mechanisms include fork stabilization, lesion bypass, template switching through recombination or fork reversal and double-strand break (DSB)-mediated restart (4, 14). SMARCAL1 is recruited to stalled replication forks through a direct interaction with RPA (15, 16), the major ssDNA-binding protein in eukaryotic cells. However, the mechanism by which SMARCAL1 promotes the repair of DSBs generated during DNA replication is unclear.

SMARCAL1 mutations are linked to Schimke immuno-osseous dysplasia (SIOD), a multisystem autosomal recessive disorder characterized by T-cell immunodeficiency, spondyloepiphyseal dysplasia, steroid-resistant nephrotic syndrome and susceptibility to cancer (17–23). Numerous SMARCAL1 gene mutations have been identified, ranging from frameshift and deletions, which generally lead to protein loss, to missense mutations that differently affect the protein’s expression, activity, stability and location (21, 24, 25). Although patients with SIOD show varying degrees of disease severity depending on the SMARCAL1 mutations, ranging from severe disease leading to death in the first years of life to mild disease with survival into adulthood, a clear genotype-to-phenotype correlation has not been defined; therefore neither the clinical expression nor the outcome can be accurately predicted (21, 26).

Loss of SMARCAL1 function in cells causes increased sensitivity to γ-IR and to several genotoxic agents inducing DSBs (7–9). In contrast, patients with SMARCAL1-deficiency exhibit reduced V(D)J recombination products in peripheral lymphocytes and increased chromosomal breakage, suggesting that SMARCAL1 is required for efficient nonhomologous end-joining (NHEJ) in human cells (27). However, its role in the homologous recombination (HR) pathway has not yet been addressed.

In this study, we employed primary and SV40-immortalized fibroblasts from a patient with SIOD who carried the homozygous c.1921dup (NM_014140.3) mutation in SMARCAL1 (28). Our objective was to investigate the role of SMARCAL1 in the two major DSB DNA repair pathways, HR and NHEJ. Our experiments using γ-H2AX foci analysis in combination with survival experiments demonstrate a considerable DSB repair defect in the S/G2 phase cell cycle in SMARCAL1-deficient fibroblasts. Moreover, the strong correlation between unrepaired DSBs and sensitivity to agents that cause oxidative damage and replication fork collapse supports the contribution of SMARCAL1-dependent DSB rejoining to survival in the S/G2 phase cell cycle, which differs from the situation in G1, representing an abrogated HR pathway. Our results suggest that SMARCAL1 is a novel factor required for efficient HR-mediated DSB repair as a result of oxidative damage and stalled replication forks.

Cell Culture

Primary and SV40-immortalized human fibroblasts and HEK-293T were cultured in a humidified 5% CO₂ atmosphere at 37 ºC in Dulbecco’s Modified Eagle’s medium (DMEM; Gibco) supplemented with 15% fetal bovine serum (FBS; Gibco) and antibiotics (amphotericin, streptomycin and penicillin; Gibco).

Treatment with DNA- damaging agents

Irradiation was carried out using a ¹³⁷Cs source at a dose rate of 8 Gy/min (Gammacell® 1000 Elite MDS Nordion)., hydroxyurea (HU), H₂O₂ and Etoposide were purchased from Sigma-Aldrich (Poole, UK).

Immunofluorescence Analysis

Cells were fixed in 4% paraformaldehyde, 2% sucrose phosphate-buffered saline (PBS) for 10 min at room temperature (RT) and permeabilized in PBS/2% Triton X-100 (Sigma, St. Louis, US) for 5 min at RT. Coverslips were washed in PBS prior to immunostaining. Primary antibody incubations were performed for 30 min at 37ºC (1:800 for γ-H2AX, Abcam) and overnight at 4ºC (1:500 for 53BP1, BD Biosciences, USA and Rad51) in PBS supplemented with 2% bovine serum fraction V albumin (BSA) and followed by washing in PBS. Incubations with Alexa Fluor 488-conjugated secondary antibody (1:800, Molecular Probes) were performed at 37 ºC in 2% BSA for 30 min at RT. For 53BP1 and Rad51 staining, cells were blocked for 60 min in Tris-buffered saline, 0.1% Tween 20 (TBST)/5% BSA before primary antibody incubations. For Rad51 staining, fibroblasts were extracted with the extraction buffer (20 mM HEPES, pH 7.0; 100 mM NaCl; 300 mM Sucrose; 3 mM MgCl₂; 1 mM ethylene glycol tetraacetic acid (EGTA), 0.5% Triton X-100) for 10 min before fixation. Lastly, the cells were washed in PBS and coverslips were mounted in Vectashield (Vector Laboratories, Peterborough, UK) with DAPI (4,6 diamidino-2-phenylindole) (Atom), and γ-H2AX, 53BP1 and RAD51 foci were quantified using a fluorescent microscopy (Zeiss AxioImages.A1, Carl Zeiss).

Survival Assay

Primary human fibroblasts were seeded at a density of 1×10⁴ cells/mL in T75 flasks and irradiated with ionizing radiation (IR) (¹³⁷Cs) in 3 independent experiments. Eleven days later, the adherent cells were trypsinized and counted. The resulting values were set relative to non-irradiated cells.

Cell Viability Assays

MTT assay: SV40-immortalized human fibroblasts were seeded at a density of 5000–10,000 cells/well in a 96-well microplate and allowed to attach overnight. The cells were then treated with differing concentrations of H₂O₂ and HU and incubated at 37 ºC for 1 h and 18 h, respectively. The medium was then removed and replaced with 100 µL of fresh DMEM and 10 µL of MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added to each well and incubated for 4 h at 37 ºC. After the incubation, 100 µL of solubilization solution was added to each well. The microplate was incubated overnight at 37 °C. The next day, the assay was read and recorded at 500 nm, with the reference wavelength set to 690 nm using a Synergy™ 2 Multi-Mode Microplate Reader.

Clonogenic assay

Primary fibroblasts were seeded at a density of 500–1000 cells/mL in 60-mm dishes in duplicate, allowed to attach overnight and then treated with varying concentrations of H₂O₂ and HU. Cells were incubated at 37°C for 3–4 weeks. To evaluate cell survival, dishes were fixed and stained with 0.5% gentian violet (crystal violet) for 30 min at RT, then rinsed with water and left to dry at RT. The number of colonies per dish was counted with ColonyCountJ software (186), and the survival fractions were calculated as the ratio of the plating efficiencies of treated cells to untreated cells.

Cloning and Site-Directed Mutagenesis

Wild-type SMARCAL1 (NM_014140.3) was cloned between HindIII and BamHI sites of pcDNA3.1 + vector (GenScript). QuikChange II Site-Directed Kit (Agilent Technologies, USA) was employed to generate SMARCAL1 mutation (c.1920_1921 insG). Plasmid DNAs were isolated using Qiaprep Spin Miniprep kit (Qiagen). The mutagenized plasmid was then sequenced by Sanger and analyzed with SnapGene software. The following oligonucleotides were used for mutagenesis:

Forward primer: 5’-GCTGGAGGAAGCAGGTCATGCTGCGGC-3’,

Reverse primer: 5’-GCCGCAGCATGACCTGCTTCCTCCAGC-3’.

Sequencing primer: 5’- CGCAGATCATCGCAGTCAAGCCAACT-3’.

Biochemical Analysis

Plasmids were transiently transfected into primary fibroblasts and HEK-293T cells using FuGENE® HD Transfection Reagent (Promega). Twenty-four hours after the transfection, the cells were lysed and analyzed by western blot. After sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were transferred from polyacrylamide gels to polyvinylidene fluoride (PVDF) membrane by wet transfer according to Towbin (Towbin et al., 1979) and probed with SMARCAL1 antibodies (Thermo Fisher Scientific) and α-tubulin (Sigma-Aldrich, USA). Proteins of interest were detected with HRP-conjugated goat anti-mouse or anti-rabbit IgG secondary antibodies (DAKO) and visualized with Pierce ECL Plus Western Blotting Substrate (Thermo Fisher Scientific, USA). Blots were imaged using an Odyssey Infrared Imaging System and analyzed with Image Studio software (LI-Cor Biosciencies, USA).

Statistical Analysis

Mean and SE values of three independent experiments were determined. The values were normalized against the control samples and plotted on a histogram graph. The p-values were calculated using a two-tailed Student’s t-test. For this study, the following legend was adopted: n.s, non-significant, *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001.

Analysis of SMARCAL1 protein expression

We studied the effect of the c.1921dup (p.V641GfsX51) mutation on SMARCAL1 protein expression. Based on the domain organization of the SMARCAL1 protein, it can be predicted that this mutation can lead to a truncated protein without the C-terminal helicase region that helps to stabilize stalled replication forks (Fig. 1a). Given that frameshift mutations have been reported to generally cause a loss of detectable SMARCAL1 protein (21), immunoblots of whole-cell lysates from control and patient fibroblasts were performed to analyze wild type (wt) and mutant (mt) SMARCAL1 protein expression. To this end, a SMARCAL1 antibody raised against a region within amino acids 120 and 457 of human SMARCAL1 protein was employed. None of the SMARCAL1 proteins, wt (110 kDa) and truncated (76 kDa, predicted), were observed in the patient’s fibroblasts (Fig. 1b). However, truncated SMARCAL1 protein was expressed at high levels in transfected HeLa cells (Fig. 1b). We cannot rule out that small amounts of truncated SMARCAL1 protein might be expressed below the detection limit of Western blotting in SMARCAL1-deficient fibroblasts.

Normal DSB repair in SMARCAL1-deficient fibroblasts after doses of low ionizing radiation

T-cell immunodeficiency is a typical feature of patients with SIOD and can be explained by a defective recombination process involving DNA recombination, DNA damage recognition, and subsequent DNA damage repair through the NHEJ pathway, which would also explain the restricted T-cell receptor repertoire expressed by the peripheral T cells from these patients (28)). To further investigate the role of SMARCAL1 in the NHEJ pathway, we observed DSB repair kinetics by scoring the in situ modification of the histone variant H2AX (γ-H2AX), which is phosphorylated proximal to DSB sites (31). We employed non-replicating G0/G1 phase cells to avoid γ-H2AX foci formation at stalled replication forks. An analysis of the time course for DSB repair by γ-H2AX showed no differences in DSB repair after administering ionizing radiation (Fig. 2a and c). However, slight differences were observed at 1 h post-IR.

It has been reported that the NHEJ pathway also plays a dominant role in repairing DSBs caused by chemotherapeutic topoisomerase II inhibitors, such as ICRF193 and etoposide (32). We therefore investigated γ-H2AX focus formation at several time points after treatment with etoposide. Following induction of DNA damage, SMARCAL1-deficient fibroblasts displayed the same number of γ-H2AX foci 24 h after γ-IR than control fibroblasts (Fig. 2b and d).

Taken together, these results indicate that DNA breaks generated in the G1 phase of the cell cycle are efficiently repaired in the absence of SMARCAL1. Therefore, a defect in the NHEJ pathway, responsible for the DSB repair in the G1 phase, would not explain the T lymphopenia observed in our patient with SIOD.

We have previously shown that SMARCAL1-deficient and control fibroblasts exhibited comparable amounts of γ-H2AX foci at 24 h post-IR (Fig. 2a and c), suggesting that the absence of SMARCAL1 does not result in increased cellular sensitivity to γ-IR. To corroborate this finding, we assessed the relative survival of the cells after exposure to γ-IR. Cell survival experiments showed that SMARCAL1-deficient fibroblasts exhibited increased radiosensitivity, and the magnitude of this radiosensitivity was similar to that shown by cells from patients with mutations in proteins involved in the NHEJ repair pathway such as Artemis and ligase IV (Fig. 2e). Our results therefore show that although the γ-IR-induced DSB repair was not affected in SMARCAL1-deficient fibroblasts, their viability was decreased after γ-IR exposure. We propose that the decreased survival observed in SMARCAL1-deficient cells is consistent with the level of unrepaired DSBs observed in asynchronous SMARCAL1-deficient cells where the HR pathway plays a key role (see next section).

SMARCAL1 plays an important role in the DNA damage response through the homologous recombination pathway

Several published studies have suggested a role for SMARCAL1 at sites of DNA DSBs following stress replication or oxidative damage (33, 34). It has been reported that DSBs can arise spontaneously during DNA replication following oxidative stress and that HR is the main pathway responsible for repairing this type of DNA damage. We therefore aimed to evaluate the role of SMARCAL1 in repairing DSBs resulting from oxidative damage and stalled replication forks. Asynchronous SMARCAL1-deficient fibroblasts were exposed to 1 M H₂O₂ for 30 min, and γ-H2AX foci were evaluated at different times after treatment. The analysis of the time course for DSB repair by γ-H2AX assay showed a pronounced repair defect in SMARCAL1-deficient fibroblasts, as shown by the high number of γ-H2AX foci that remained 6 and 24 h after treatment (Fig. 3a and d). We also performed the same experiment using low doses of H₂O₂ to simulate the concentrations present in the cell nucleus. When we used a dose of 200 µM H₂O₂, SMARCAL1-deficient fibroblasts showed a delay in the DNA repair kinetics at 6 and 24 h post-treatment (Fig. 3b and e) suggesting an essential role for SMARCAL1 in the DSB repair through the HR pathway.

Given that γ-H2AX foci has been shown to form independently of the generation of DSBs, 53BP1 staining was also used as a marker of DSBs. 53BP1 is a key factor in DNA damage response and plays a key role in determining the choice of DSB repair pathway in the G1, S, and G2 phases of the cell cycle (35)). Thus, SMARCAL1-deficient fibroblasts were exposed to 200 µM H₂O₂ for 1 h, and 53BP1 nuclear foci were evaluated at different times after treatment. The analysis of the time course for DSB repair by 53BP1 foci assay also showed defective DSBs repair in SMARCAL1-deficient cells, as shown by the higher number of 53BP1 foci present 24 h after post-irradiation (Fig. 3c and f).

Several studies have described SMARCAL1 as a replication stress response protein that maintains genome integrity at stalled replication forks (34). Consistent with the proposed role of SMARCAL1 to overcome DNA replication stres, this type of DNA damage response was analyzed by quantifying the number of γ-H2AX foci at different time points after treatment with 0.5 mM HU. Treatment of cells with HU leads to stalled replication forks and the creation of repair foci that include RPA and γ-H2AX. The results showed that 2 h after treatment, SMARCAL1-deficient fibroblasts accumulate more γ-H2AX foci than control fibroblasts. Furthermore, the repair kinetics of DSBs was delayed in SMARCAL1-deficient cells, indicated by the higher number of γ-H2AX foci at 24 h post-treatment (Fig. 4a and b). We can therefore conclude that SMARCAL1-deficient fibroblasts displayed increased DNA damage and defective DSB repair after treatment with HU, demonstrating a requirement of SMARCAL1 to respond to multiple forms of replication stress. Lasty, to determine whether the lack of SMARCAL1 enhances RAD51 association with stalled replication forks, RAD51 foci were quantified in SMARCAL1-deficient fibroblasts exposed to 4 mM HU. For repair times of 2 h and longer, SMARCAL1-deficient cells showed significantly more RAD51 foci than the control cells (Supplemental Fig. S1). Taken together, these results show that cells lacking SMARCAL1 have a defect in their response to HU-induced DNA damage, suggesting that a larger amount of DSBs must be repaired through the HR pathway.

SMARCAL1-deficient fibroblasts are sensitive to oxidative damage and replication stress

To define the role played by SMARCAL1 in cell sensitivity to several DSB-inducing agents, we measured the cellular response to several DNA-damaging agents. Cell viability and proliferation were determined by MTT and clonogenic assays, respectively.

To investigate whether SMARCAL1-deficiency results in sensitivity to oxidative damage, we measured the clonogenic survival of cells treated with various concentrations of H₂O₂. As shown in Fig. 5a and b, H₂O₂ doses within the 1–50 µM range, representing the local H₂O₂ concentrations in the nucleus, produced minimal cell death in control and SMARCAL1-deficient fibroblasts. However, at doses higher than 50 µM, SMARCAL1-deficient fibroblasts showed increased sensitivity relative to control fibroblasts. The MTT assay also showed that SMARCAL-1 deficient cells had reduced survival rates (Fig. 5c), but significant differences were only observed at higher doses (600–1200 µM).

SMARCAL1 activity has been reported to be essential for proper nucleic acid metabolism at sites of replication stress; therefore, cells lacking SMARCAL1 should be hypersensitive to replication stress agents. To demonstrate this hypothesis, we determined the relative survival rate of control and SMARCAL1-deficient fibroblasts after treatment with HU. The clonogenic assay showed that survival was decreased in SMARCAL1-deficient fibroblasts; however, this result was observed only at concentrations greater than 150 µM (Fig. 6a and b). The MTT assay thereby also showed hypersensitivity of SMARCAL1-deficient fibroblasts after HU treatment, yielding significant differences at 100 µM (Fig. 6c). These results are consistent with those reported in previous studies and corroborate the assertion that SMARCAL1 is crucial for maintaining cell viability after exposure to genotoxic agents that induce DSBs at stalled replication forks.

The SMARCAL1 frameshift mutation is associated with the severe form of SIOD and predisposition to cancer development

The SMARCAL1 frameshift mutation (28) is located at the HARP2-ATPase catalytic domain and leads to a truncated protein that is not expressed in the patient’s fibroblasts. This mutation has been previously described in a compound heterozygous patient with SIOD who presented with spondyloepiphyseal dysplasia, nephrotic syndrome with progressive renal failure and cerebellar disorder (17, 24). Although this frameshift mutation has been associated with severe forms of SIOD, neurological manifestations were not observed in any of the patients. As demonstrated in this study, the p.V641Gfs*51 mutation adversely affects protein function, leading to a severe clinical course for the patient, who did not survive beyond the age of 13 years.

SIOD has been associated with several malignancies, including non-Hodgkin lymphoma and osteosarcoma (36), mainly due to SMARCAL1 activity, which is required to prevent the accumulation of DNA damage during DNA replication (6, 37, 38). It is therefore possible that the susceptibility to cancer observed in certain patients might depend on the specific mutation and degree of functional activity. Patients with non-sense or frameshift SMARCAL1 mutations could be more susceptible to developing cancer than those patients with missense mutations allowing partial SMARCAL1 function.

DNA replication stress is prevalent in human cancers but absent in normal cells, suggesting that the proteins involved in the cellular response to DNA replication stress could be potential therapeutic targets for cancers with defects in DNA damage repair and cell cycle checkpoints. It has been reported that mice lacking SMARCAL1 activity have delayed development of MYC-induced B-cell lymphomas (39). Also, Smarcal 1 knockdown showed increased sensitivity of gastric cancer cell lines to DNA damaging agents.

T-cell lymphopenia observed in patients with SIOD is not associated with a defect in the NHEJ pathway

The molecular mechanisms underlying the severe lymphocytopenia of patients with SIOD remain unclear. SMARCAL1 has been reported to be involved in the NHEJ repair process of DSBs (27), which could explain the T-cell immunodeficiency presented by these patients. However, the functional analysis of the NHEJ repair pathway performed in this study indicates that SMARCAL1-deficient fibroblasts are proficient in DSB repair in the G1 phase, and therefore a defect in this pathway might not explain the T lymphopenia observed in these patients. These findings agree with the published results that report that SMARCAL1 is recruited to sites of DNA damage only during the S and G2 phases where the main repair pathway is HR (12, 37). Data from the replication stress response after HU treatment might offer an explanation behind the immunodeficiency exhibited by patients with SIOD. Our results indicate that the patient’s fibroblasts showed decreased cell viability and a defective DSB response to HU, an exogenous DNA damage agent that induces DNA replication stress. Given that hematopoietic cells proliferate rapidly, they are exposed to a threat to genomic integrity through replication stress. The loss of SMARCAL1, an important protein for maintaining the survival and function of hematopoietic cells under DNA replication stress conditions, could therefore explain the lymphopenia reported in this patient. This hypothesis has not been directly addressed in patients with SIOD, but certain results in Smarcal1-deficient mice strongly point to a hematopoietic progenitor cell defect (40). We cannot absolutely consider these factors as the sole cause of T-cell deficiency because it has been observed that the lack of interleukin (IL)7Rα expression in T cells is a hallmark of T-cell immunodeficiency in patients with SIOD (40). IL-7 produced by thymic stromal cells is essential for T-cell development and signaling through the IL-7 receptor complex. Both complete or partial deficiency of IL7Rα abrogates T-cell development and causes severe T-cell immunodeficiency (42).

In summary, we propose that the T lymphopenia of patients with SIOD is mainly attributable to the significantly reduced cell viability together with attenuated fork replication stabilization in which SMARCAL1 plays a key role.

SMARCAL1 participates in DSB repair through the homologous recombination HR pathway

By using γ-H2AX foci analysis as an approach for DSB repair measurements, we conducted the first study of DSB repair in human SMARCAL1-deficient fibroblasts using genotoxic agents inducing oxidative damage and replication stress. We have shown that after induction of replication stress by HU, SMARCAL1-deficient cells held in the S/G2 phase have a significant repair defect, indicating that a significant fraction of the induced breaks remains unrepaired 24 h after treatment. Furthermore, Rad51 foci analysis also show a delayed DNA repair kinetic in SMARCAL1-deficient cells suggesting that the absence of SMARCAL1 enhances Rad51 association with stalled replication forks, indicating a collapse in the replication forks as a consequence of a greater number of DNA breaks that need the HR pathway to be repaired. These findings agree with previous studies that show that SMARCAL1 depletion leads to an increased frequency of Rad51 foci in HU-treated cells (43) and support the role of SMARCAL1 in DSB repair through the HR pathway. We also analyzed the sensitivity of these cells to HU using clonogenic and MTT assays, demonstrating that the loss of SMARCAL1 causes hypersensitivity to replication stress, as has been previously shown in other studies (8, 44). The correlation between unrepaired DSBs and survival in these experiments suggests that the striking sensitivity observed in SMARCAL1-deficient cells can be attributed to a defect in repairing a subset of DSBs generated during the G2 phase cell cycle.

We have also shown that SMARCAL1-deficient fibroblasts treated with H₂O₂ display a significant increase in γ-H2AX and 53BP1 foci, indicating a high number of DSBs that must be repaired through the HR pathway. Therefore, in the absence of SMARCAL1, the HR pathway does not work properly, and breaks remain unrepaired for longer. These results are consistent with those of Bansal et al. who showed that cells from patients with SIOD presented deficiency in DNA damage response after oxidative stress, as evidenced by increased γ-H2AX foci, which can be restored by overexpression of the SMARCAL1 protein. Survival experiments performed in SMARCAL1-deficient cells showed reduced viability after H₂O₂ treatment. These results therefore suggest that loss of SMARCAL1 confers sensitivity to H₂O₂ treatment by accumulating oxidative damage and DSBs in the S-phase, with a high percentage of cells undergoing apoptosis due to attenuation of DNA damage repair.

The most significant finding of this study is that the absence of SMARCAL1 increases cell sensitivity to oxidative DNA damage. Our hypothesis is that certain oxidative DNA lesions persists during the S-phase, where they produce DSBs that are more cytotoxic and require repair via replication-linked HR, rather than base excision repair. This reliance on the HR pathway to recover from oxidative DNA lesions might account for the accumulation of certain types of oxidative lesions in SMARCAL1-deficient cells, leading to genome instability and predisposition to developing certain types of tumors.

In summary, we present cellular and biochemical evidence that the SMARCAL1 c.1920_1921insG frameshift mutation impairs SMARCAL1 protein expression, causing defects in DNA damage response and hypersensitivity to several genotoxic agents that cause DSBs and replication fork collapse. The latter data confirm the hypersensitivity to DNA-damaging agents in vivo and provide guidance for managing patients with SIOD.

Competing Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Ethics approval

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by CEIm Hospital Clinico San Carlos.

Consent to participate

Written informed consent was obtained from the parents.

Funding Information

This work was supported by grants from the Ministerio de Economía y Competitividad (MINECO SAF2014-54708-R), Instituto de Salud Carlos III (ISCIII) through the project PI17/00543, PI22/00790 and Fundación para la Investigación Biomédica del Hospital Universitario La Paz (FIBHULP) through grants Luis Álvarez 2022. PPC was supported by Juan de la Cierva-Incorporación fellowship (IJCI-2014-19262).

Author Contribution

P.P.C. Experiments, data analysis, manuscript comments and editingA.S. immunofluorescence experiments, survival and cell viability assaysM.S.P. and R.C.G. Cloning, site-directed mutagenesis and protein expressionE.R.O. Physician in charge of the patient’s careS.S.R. and R.P.D. Manuscript comments, advice and editingM.J.R. Experiment design, manuscript drafting and editing. Corresponding author.All authors contributed to the article, reviewed the manuscript and approved the submitted version.

Acknowledgments

We would like to thank the patient and his family for participating in this study.

Data Availability

The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.

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No competing interests reported.

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Figure S1. Rad51 foci formation after HU-induced DSBs in SMARCAL1-deficient fibroblasts. a Primary fibroblasts from the SMARCAL1-deficient patient (SMARCAL1-/-) and a healthy control were treated with 4 mM HU for 18 h and fixed at the indicated times after treatment. DNA damage foci were detected using antibodies against Rad51. bHistogram representing the mean number of Rad51 foci per cell. Error bars throughout this figure depict the mean and standard deviation from one single experiment where >30 cells were scored for each time point.

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SMARCAL1: a DNA repair protein recruited to double-strand breaks at stalled replication forks to promote genome stability through a homologous recombination pathway

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Version 1

Abstract

Figures

INTRODUCTION

MATERIALS AND METHODS

Cell Culture

Immunofluorescence Analysis

Survival Assay

Cell Viability Assays

Clonogenic assay

Cloning and Site-Directed Mutagenesis

Biochemical Analysis

Statistical Analysis

RESULTS

Analysis of SMARCAL1 protein expression

Normal DSB repair in SMARCAL1-deficient fibroblasts after doses of low ionizing radiation

SMARCAL1 plays an important role in the DNA damage response through the homologous recombination pathway

SMARCAL1-deficient fibroblasts are sensitive to oxidative damage and replication stress

DISCUSSION

SMARCAL1 participates in DSB repair through the homologous recombination HR pathway

CONCLUSION

Declarations

Competing Interest

Ethics approval

Funding Information

Author Contribution

Acknowledgments

Data Availability

References

Additional Declarations

Supplementary Files

Status:

Version 1