The SIOD disorder protein SMARCAL1 is an RPA-interacting protein involved in replication fork restart

Abstract

The integrity of genomic DNA is continuously challenged by the presence of DNA base lesions or DNA strand breaks. Here we report the identification of a new DNA damage response protein, SMARCAL1 (SWI/SNF-related, matrix associated, actin-dependent regulator of chromatin, subfamily a-like 1), which is a member of the SNF2 family and is mutated in Schimke immunoosseous dysplasia (SIOD). We demonstrate that SMARCAL1 directly interacts with Replication protein A (RPA) and is recruited to sites of DNA damage in an RPA-dependent manner. SMARCAL1-depleted cells display sensitivity to DNA-damaging agents that induce replication fork collapse, and exhibit slower fork recovery and delayed entry into mitosis following S-phase arrest. Furthermore, SIOD patient fibroblasts reconstituted with SMARCAL1 exhibit faster cell cycle progression after S-phase arrest. Thus, the symptoms of SIOD may be caused, at least in part, by defects in the cellular response to DNA replication stress.

Figure 1.

Interaction between SMARCAL1 and RPA in human cell lines. (A) Input cell lysates and HA immunoprecipitates from 293T-Rex cells expressing control HA vector, HA-RPA1, or HA-SMARCAL1 were immunoblotted with antibodies to HA, SMARCAL1, RPA1, and RPA2. Cells were treated with 10 Gy IR or 30 J/m² UV or left untreated. (B) Protein complexes immunoprecipitated with IgG or anti-SMARCAL1 antibodies from 293T-Rex were analyzed for SMARCAL1 and RPA2 by Western blotting. (C) Protein complexes immunoprecipitated from 293T-Rex cells with IgG or anti-RPA2 antibodies were immunoblotted with antibodies to SMARCAL1, RPA1, and RPA2.

Figure 2.

In vitro interaction between SMARCAL1 and RPA. (A) Western blot of protein complexes pulled down with nickel beads from BL21 (DE3) bacteria expressing untagged RPA1, RPA2, or RPA3, with or without His-tagged SMARCAL1. (B) Sequence alignment of the putative RPA2-interacting motif of SMARCAL1 with the previously known RPA2-interacting motifs of TIPIN, UNG2, XPA, and RAD52. Similar residues are indicated within boxes. Sequence alignments were performed with ClustalW. The schematic representation of human SMARCAL1 and the RQK and ΔN mutants is shown below the sequence alignments. The amino acids mutated to alanine in the RQK mutant are indicated by asterisks. (C) Western blot of protein complexes pulled down from bacteria coexpressing RPA1, RPA2, and RPA3 with nickel beads in the presence of either wild-type, RQK, or ΔN His-tagged SMARCAL1. (D) HA immunoprecipitates from 293T-Rex cells expressing wild-type, RQK, or ΔN HA-SMARCAL1 were immunoblotted with antibodies to HA and RPA2.

Figure 3.

Colocalization of SMARCAL1 and RPA at DNA damage sites. (A) U2OS cells expressing GFP-SMARCAL1 were stained with anti-GFP (green) and anti-RPA2 (red) antibodies following treatment with 2 mM HU, 10 nM CPT, or microirradiation with UV laser. (B) GFP-SMARCAL1 U2OS cells transfected with control or CtIP siRNAs were stained with anti-GFP (green) and anti-γH2AX (red) antibodies following UV laser microirradiation. Merged images have DAPI staining (blue). (C) Percentage of control or CtIP siRNA-treated U2OS cells displaying GFP-SMARCAL1 or GFP-RPA1 stripes over the total number of cells with γH2AX stripes following UV laser microirradiation. The data represent the average of two independent experiments. (D) Cell lysates from cells described in C were immunoblotted for CtIP and GAPDH. (E) U2OS cells expressing wild-type and mutant GFP-SMARCAL1 constructs were microirradiated with a UV laser and stained as in B.

Figure 4.

DNA damage sensitivity of SMARCAL1-depleted cells. (A) Cell competition assay showing survival of U2OS cells infected with SMARCAL1 shRNA or FF shRNA retroviruses, relative to dsRed-expressing U2OS cells, following treatment with DNA-damaging agents. (IR) 5 Gy IR; (UV) 7 J/m² UV; (MMC) 150 nM MMC; (CPT) 10 nM CPT. (B) Cell competition assay using siRNAs targeting SMARCAL1, ATM, or ATR, or a nontargeting control siRNA. Treatments are the same as described in A. (C) Western blot showing depletion of SMARCAL1 in cells infected with retrovirus expressing SMARCAL1 shRNA. (D) Western blot showing knockdown of SMARCAL1 in U2OS cells transfected with four individual SMARCAL1 siRNAs.

Figure 5.

Effects of SMARCAL1 depletion on cell cycle progression. (A) Analysis of FF control and SMARCAL1-depleted U2OS cells 10 h after a BrdU pulse alone (not treated, NT), or a BrdU pulse followed immediately by IR (2 Gy) or addition of CPT (5 nM). Nocodazole was added after the BrdU pulse to capture mitotic cells, which are measured by staining for P-H3. Shown are the percentages of BrdU⁺ cells that are also P-H3⁺. Values are means and standard deviations from at least two experiments. P-values were calculated using a one-tailed t-test. (B) Analysis of FF control and SMARCAL1-depleted U2OS cells 10 h after a BrdU pulse followed by 2 Gy IR or addition of 5 nM CPT in the presence of 5 mM caffeine. The analysis was performed as described in A. The numbers indicate the percentage of BrdU⁺ P-H3⁺ cells. (C) Analysis of mitotic cells (P-H3⁺) 10 h after release from the thymidine block. Propidium iodide was used to measure DNA content. Numbers indicate the percentage of P-H3⁺ cells.

Figure 6.

Effects of SMARCAL1 depletion on replication fork restart. (A) Restart of DNA replication in FF control or SMARCAL1 shRNA-depleted U2OS was measured by incorporation of BrdU following release from the thymidine block. BrdU (10 μM) was added 30 min prior to fixation. 7-AAD was used to measure DNA content. The percentage of BrdU⁺ cells is indicated. Results are representative of three independent experiments. (B) Schematics of the pulse-labeling experiment for DNA fiber analysis. (C) DNA fibers were stained with antibodies recognizing IdU (green) or CldU (red). Anti-ssDNA antibody was used as a control. (D) Analysis of the percentage of stalled or collapsed replication forks (green tracts only) in FF control or SMARCAL1 shRNA-depleted U2OS cells with or without aphidicolin treatment. The percentage of new origins fired during CldU treatment (red tracts only) is also shown.

Figure 7.

Analysis of cell cycle progression in SIOD patient fibroblasts reconstituted with SMARCAL1. (A) DNA replication restart in hTERT-immortalized SIOD patient SD31 fibroblasts and SD31 fibroblasts reconstituted with untagged SMARCAL1 was measured by incorporation of BrdU 3 h after release from a thymidine block. Results are means and standard deviations of two independent experiments. P-value was calculated using a two-tailed t-test. (B) Analysis of cell cycle progression in hTERT-immortalized SD31 fibroblasts and SD31 fibroblasts reconstituted with untagged wild-type or mutant SMARCAL1 constructs following a thymidine block. The graph shows the fold change in P-H3⁺ cells relative to nonreconstituted hTERT SD31 fibroblasts 15 h after release from a thymidine block into nocodazole. Values are means and standard deviations from at least two experiments. (*) P = 0.003. The P-value was calculated using a two-tailed t-test. (C) Western blot showing the expression of wild-type and mutant SMARCAL1 protein in the reconstituted hTERT-immortalized SD31 fibroblasts.