PMC

The SMARCAL1 RPA32-binding domain is not necessary for ATPase or annealing helicase activities in vitro. (A) Coomassie-stained gel of the Flag-SMARCAL1 and Flag-SMARCAL1-Δ32 purified from baculovirus-infected insect cells. (B) ATPase activity of the wild-type and Δ32 SMARCAL1 proteins was measured in the presence of the indicated concentrations of fork DNA. Error bars are standard deviation (n = 3). (C,D) Annealing helicase activities of wild-type and Δ32 SMARCAL1 proteins. The concentration of SMARCAL1 protein in C is 300 nM.

Deregulation of SMARCAL1 expression causes activation of the DNA damage response. (A) U2OS cells were transfected with siRNA targeting SMARCAL1 or expression vectors encoding either SMARCAL1 or GFP-SMARCAL1. Three days after transfection, cells were stained with antibodies to γH2AX and appropriate secondary antibodies. The percentage of cells with γH2AX staining was scored. Error bars are standard deviation (n ≥ 3). (*) P < 0.05. (B) Representative images of γH2AX staining. (C) Immunoblots of U2OS cell lysates with antibodies to SMARCAL1 or CHK1 after transfection with the indicated SMARCAL1 siRNAs. (NT) Nontargeting.

RPA binding and annealing helicase activities of SMARCAL1 are required for its cellular functions. (A) HeLa cells were transfected with expression vectors (pLEGFP backbone for high levels of expression) encoding GFP-tagged wild-type SMARCAL1 (WT), the annealing helicase-deficient SIOD patient mutant protein R764Q, or SMARCAL1-Δ32. The amount of DNA damage response activation was scored by monitoring KAP1 phosphorylation. (B) Expression levels of the GFP-SMARCAL1 proteins were determined by immunoblotting. The loading control was either ATM or RPA32. (C) The percentage of HU-treated cells in which GFP-SMARCAL1 wild-type or mutant proteins localized to intranuclear foci was scored. Cells were treated with HU for 8 h. (D) U2OS cells were cotransfected with the indicated pLL5.0-GFP-GW plasmids that encode siRNA-resistant SMARCAL1 cDNAs and siRNA targeting SMARCAL1. GFP-positive cells were scored for γH2AX staining. (GFP) GFP empty vector; (NT) nontargeting siRNA control. Error bars in all panels are standard deviation (n = 6).

ATM, ATR, and DNA-PK phosphorylate SMARCAL1. (A) Cell lysates from cells treated with 1 mM HU, 10 Gy of IR, or 50 J/m² of UV radiation were separated by SDS-PAGE and immunoblotted. The time points represent the duration of the HU treatment or the length of time the cells were allowed to recover following the radiation exposure. (Unt) Untreated. (B) Cell lysates from untreated or HU-treated cells were incubated in the presence of λ phosphatase and the phosphatase inhibitor sodium vanadate as indicated for 30 or 60 min prior to separation by SDS-PAGE and immunoblotting. (C,D) Flag-ATM or Flag-ATR wild-type (WT) or kinase-dead (KD) proteins or wild-type Flag-DNA-PKcs were immunopurified from transfected cells. (Right panel) Wild-type DNA-PK was also purchased from Promega. The kinases were incubated with purified SMARCAL1 in the presence of γ-³²P-ATP. Sonicated salmon sperm DNA was added to the DNA-PK reactions as indicated. Reactions were separated by SDS-PAGE and subjected to autoradiography (³²P). The levels of SMARCAL1, ATM, ATR, and DNA-PKcs proteins in each reaction were visualized by Coomassie staining. (E) 293T cells were transfected with either nontargeting (NT) or ATR siRNA. Cells were also treated with specific ATM (KU55933, 10 μM) () or DNA-PK (KU57788, 1 μM) () inhibitors as indicated and exposed to 1 mM HU or 10 Gy of IR. Cell lysates were separated by SDS-PAGE and immunoblotted for SMARCAL1 or ATR.

SMARCAL1 functions at stalled replication forks. (A,B) U2OS cells were transfected with a SMARCAL1 expression vector then fixed and stained with the indicated antibodies and appropriate fluorophore-conjugated secondary antibodies. In A, the cells were pulsed with BrdU for 20 min prior to staining. (C,D) U2OS cells transfected with siRNA targeting CHK1 or SMARCAL1 were stained with antibodies to γH2AX and cyclin A. Cells were scored as positive for one or both proteins by immunofluorescence imaging. Nontransfected cells were also irradiated with 5 Gy of IR for comparison. (E–G) U2OS cells were transfected with a GFP-SMARCAL1 expression vector, then fixed and stained. Staining in F is as follows: (Panels 1–4) SMARCAL1 (green) and BrdU (red); (panels 5–8) SMARCAL1 (green) and RPA (red); (panels 9–12) SMARCAL1 (green) and PCNA (red). Panels 3, 7, and 11 are merged images, and panels 4, 8, and 12 were stained with DAPI. In G, the cells containing SMARCAL1 localized to foci were scored after addition of 2 mM HU. (H) Localization of endogenous SMARCAL1 was examined by indirect immunofluorescence with affinity-purified anti-SMARCAL1 antibody in HU or UV radiation-treated U2OS cells. These cells also stably express HA-ATRIP, which permitted analysis of colocalization using anti-HA monoclonal antibody. Specificity of the SMARCAL1 antibody was confirmed in SMARCAL1-silenced cells. (Unt) Untreated. Cells were extracted with Triton X-100 prior to staining, so only chromatin-bound SMARCAL1 is observed. (I) The percentage of cells containing endogenous SMARCAL1 in foci was scored in cells treated with HU for the indicated times. Error bars in all graphs are standard deviation (n = 3).

SMARCAL1-deficient cells exhibit increased RPA-loading onto chromatin and persistent RPA phosphorylation after replication stress, and are hypersensitive to replication stress agents. (A) The chromatin fraction of cells transfected with nontargeting (NT) or SMARCAL1 (S6) siRNA was isolated and immunoblotted with the indicated antibodies. Quantitation of the amount of RPA versus ORC2 in the chromatin fraction was performed with an Odyssey instrument (arbitrary units). (B) Cells transfected with nontargeting or SMARCAL1 siRNA were treated with HU for 5 h (HU) then the HU was removed and the cells were allowed to recover for 4 h (+4h). Total cell lysates were prepared and immunoblotted with the indicated antibodies. (Unt) Untreated. (C) Cells transfected with the indicated siRNA were split into four plates, two of which were treated with 2 mM HU for 24 h. The HU was removed and replaced with standard growth media for an additional 24 h before cell viability was quantitated using the WST-1 cell proliferation reagent (Roche). Reported values are the mean and standard deviation of three experiments. All four SMARCAL1 siRNAs yielded significant hypersensitivity to HU with P values ranging from 0.02 to 10⁻⁴. (D,E) Cells transfected with the indicated siRNAs were treated with 5 μM aphidicolin or 150 nM camptothecin and cell viability compared with untreated controls. The P value of nontargeting compared with SMARCAL1-silenced for both aphidicolin (n = 12) and camptothecin (n = 24) treatments are <10⁻⁵.

An interaction with RPA is necessary and sufficient to localize SMARCAL1 to stalled replication forks. (A) Endogenous SMARCAL1 or Flag-HA-SMARCAL1 was immunopurified from nuclear cell extracts and the resulting protein complexes were analyzed by mass spectrometry. The table indicates the number of peptides identified for each RPA protein subunit. (IgG) Control immunoprecipitation; (Sm) =Flag-HA-SMARCAL1; (Ctrl) Flag-HA empty vector cells. Where indicated, the cells were treated with 1 mM HU for 16 h prior to the purification. (B) A schematic diagram of RPA subunits. The lines below RPA32 indicate the sizes (with amino acid numbers) of the different RPA32 protein fragments identified in the unbiased two-hybrid screen using full-length SMARCAL1 as a bait. (C) HeLa cell nuclear extracts from untreated cells or cells treated with HU for 8 h were used for immunoprecipitation with anti-SMARCAL1 or preimmune antibodies. In the left panel, the lysates were treated with benzonase nuclease (Benz.) as indicated to ensure the interaction is not dependent on DNA. Immunoprecipitated proteins were separated by SDS-PAGE and immunoblotted with RPA or SMARCAL1 antibodies. (D) Sequence alignment of human, mouse, and Xenopus SMARCAL1 with human TIPIN. (E) HEK293 cells were transfected with wild-type GFP-SMARCAL1 (WT) or GFP-SMARCAL1 lacking the first 32 amino acids (Δ32). Cells transfected with an empty vector (VEC) were also prepared as a control. Anti-GFP or anti-RPA immunoprecipitated proteins were separated by SDS-PAGE and blotted with anti-GFP or anti-RPA2 antibodies. (F) U2OS cells were transfected with GFP-SMARCAL1-WT or GFP-SMARCAL1-Δ32 vectors and treated with HU for 6 h. Approximately 30% of the wild-type SMARCAL1-expressing cells had SMARCAL1 foci, while we never observed the SMARCAL1-Δ32 protein in foci. Representative images are shown. (G) GFP-SMARCAL1 containing only the first 32 or 115 amino acids of SMARCAL1 was transfected into cells, immunoprecipitated, and immunoblotted as in E. (H) The indicated expression vectors (pLL5.0-GFP-GW backbone for attenuated expression levels) were transfected into HeLa cells. The cells were treated for 7 h with 1 mM HU or left untreated and the percentage of cells containing foci of the indicated proteins was scored. Error bars are standard deviation (n = 3). (I) Representative images of the localization of SMARCAL1-1-115 and wild-type SMARCAL1.