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. 2015 Jul 27;43(13):6359-72.
doi: 10.1093/nar/gkv621. Epub 2015 Jun 18.

Smarcal1 promotes double-strand-break repair by nonhomologous end-joining

Islam Shamima Keka  1 , Mohiuddin  1 , Yuko Maede  1 , Md Maminur Rahman  1 , Tetsushi Sakuma  2 , Masamitsu Honma  3 , Takashi Yamamoto  2 , Shunichi Takeda  1 , Hiroyuki Sasanuma  4
Affiliations

Smarcal1 promotes double-strand-break repair by nonhomologous end-joining

Islam Shamima Keka et al. Nucleic Acids Res. .

Abstract

Smarcal1 is a SWI/SNF-family protein with an ATPase domain involved in DNA-annealing activities and a binding site for the RPA single-strand-DNA-binding protein. Although the role played by Smarcal1 in the maintenance of replication forks has been established, it remains unknown whether Smarcal1 contributes to genomic DNA maintenance outside of the S phase. We disrupted the SMARCAL1 gene in both the chicken DT40 and the human TK6 B cell lines. The resulting SMARCAL1(-/-) clones exhibited sensitivity to chemotherapeutic topoisomerase 2 inhibitors, just as nonhomologous end-joining (NHEJ) null-deficient cells do. SMARCAL1(-/-) cells also exhibited an increase in radiosensitivity in the G1 phase. Moreover, the loss of Smarcal1 in NHEJ null-deficient cells does not further increase their radiosensitivity. These results demonstrate that Smarcal1 is required for efficient NHEJ-mediated DSB repair. Both inactivation of the ATPase domain and deletion of the RPA-binding site cause the same phenotype as does null-mutation of Smarcal1, suggesting that Smarcal1 enhances NHEJ, presumably by interacting with RPA at unwound single-strand sequences and then facilitating annealing at DSB ends. SMARCAL1(-/-)cells showed a poor accumulation of Ku70/DNA-PKcs and XRCC4 at DNA-damage sites. We propose that Smarcal1 maintains the duplex status of DSBs to ensure proper recruitment of NHEJ factors to DSB sites.

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Figures

Figure 1.
Figure 1.
Smarcal1-deficient DT40 cells are sensitive to ICRF193 and camptothecin. Clonogenic-cell-survival assay following exposure of the indicated genotypes to DNA-damaging agents (A–D). The x-axis represents the dose of the indicated DNA-damaging agent on a linear scale; the y-axis represents the survival fraction on a logarithmic scale. Error bars show the SD of the mean for three independent assays. The P-value of (C) was calculated by Student's t-test or two-sample t-test for IR sensitivity at 2 Gy.
Figure 2.
Figure 2.
Sensitivity of human SMARCAL1−/−, SMARCAL1−/−/LIG4−/−/− and SMARCAL1−/−/DNA-PKcs−/− TK6 B cells to ICRF193 and γ-rays. (A–E) Cellular sensitivity is shown as in Figure 1. Error bars show the SD of the mean for three independent assays. P-values were calculated by Student's t-test.
Figure 3.
Figure 3.
Repair of γ-ray induced DSBs at the G1 phase in TK6 cells. (A) DNA content of G1-synchronized TK6 cells. (B) Cellular sensitivity of G1-synchronized cells to γ-rays, shown as in Figure 1. Error bars indicate the SD of the mean for three independent assays. P-value was calculated by Student's t-test. (C) Histogram representing the γH2AX subnuclear foci of G1 cells after irradiation with 1 Gy γ-rays. The x-axis represents time after γ-irradiation (time zero); the y-axis represents the average number of γH2AX foci in individual cells. The nuclei of 100 morphologically intact cells were analyzed at each time point in individual experiments. The experiment was performed at least three times, with the averages presented with SD and P-values. Asterisks indicate statistical significance; *P = 0.0055 and **P = 0.00020. (D) Histogram representing the 53BP1 subnuclear foci of G1 cells, as shown in (C). The experiment was performed at least three times, with averages presented with SD and P-value. Asterisks indicate statistical significance; *p = 6.3×10−5.
Figure 4.
Figure 4.
The loss of Smarcal1 reduces the efficiency of V(D)J recombination without compromising its fidelity. (A) The structure of two episomal V(D)J-recombination substrates, pJH200 and pJH290, and their recombination products. Open triangles and closed boxes represent recombination signals (RSs) and V(D)J-coding sequences, respectively. CamR = chloramphenicol-resistance gene; P = promoter. (B) Schematic representation of the experimental method for the V(D)J-recombination assay. Frequency of recombination was calculated by dividing the number of rearranged products (the number of camR colonies) by the number of recovered plasmids (the number of ampicillin-resistant [ampR] colonies). (C) Recombination frequency of TK6 cells carrying the indicated genotypes. Data shown are the means of more than three experiments. Error bars indicate SD of more than three independent experiments. P-value was calculated by Student's t-test. The total number of ampicillin- and chloramphenicol-resistant colonies is shown in the right panel. Supplemental Table S2 shows the nucleotide sequences of coding joints associated with deletion events.
Figure 5.
Figure 5.
The fidelity of end-joining in SMARCAL1 mutant cells. (A) Schematic diagram showing DSB-repair events that repair I-Sce1-induced DSBs in the endogenous thymidine kinase (TK) locus. TK+/− cells carry an I-Sce1 site in intronic sequences of the wild-type TK allele and a mutation in exon4 of the mutant TK allele. DSB repair associated with deletion in exon5 coding sequences would yield TK−/− clones from TK+/− cells. The number of TK−/− clones was measured by counting the number of trifluorothymidine (TFT)-resistant colonies. (B) Histogram representing the frequency of DSB-repair events (y-axis) in the indicated genotypes (x-axis). Error bars indicate SD of more than three independent experiments. P-value was calculated by Student's t-test. (C) Box plot representing the length of nucleotide deletion (y-axis) in the indicated genotypes (x-axis). PCR was performed from genomic DNA isolated from at least 50 TFT-resistant clones of each genotype, as shown in Supplementary Figure S6. (D) Schematic representation of the structure of wild-type, R764Q and Δ30 Smarcal1 proteins. SMARCAL1−/− TK6 cells were reconstituted with SMARCAL1WT, SMARCAL1Δ30 or SMARCAL1R764Q transgene. Western blot analysis for the expression of individual transgenes in SMARCAL1−/− cells. β-actin was used as a loading control. (E) Histogram representing the frequency of TFT-resistant colonies (y-axis) in the indicated genotypes (x-axis). Error bars indicate SD of more than three independent experiments.
Figure 6.
Figure 6.
The loss of Smarcal1 results in compromised accumulation of Ku70, DNA-PKcs and XRCC4 at DSB sites. (A) Western blot data showing the validation of fractionation of the cytoplasmic (Cyto), nuclear soluble (NS) and chromatin-bound (CB) fractions isolated from the whole-cell extract (WCE). (B) Western blot data show the accumulation of XRCC4 (upper panel) and Ku70 (lower panel) in the chromatin-bound fraction after one-hour exposure of cells to ICRF193. (C) Histogram showing the quantification of XRCC4 and Ku70 in (B). The y-axis represents the amount of the chromatin-bound fraction relative to the total amount of the chromatin-bound fraction plus the nuclear soluble fraction. (D) Downward arrows represent the I-Sce1- (upper) and the TALEN- (lower) cutting sites in the TK and p53 genes, respectively. Pairs of horizontal opposing arrows indicate the sets of primers for quantitative real-time PCR. (E) Histograms represent the accumulation of XRCC4 near the I-Sce1-induced DSB in the TK locus (upper panel) and near the TALEN-induced DSB at the p53 locus (lower panel). (F) Histogram represents the accumulation of Ku70 near the I-Sce1-induced DSB in the TK locus. (G) Histogram represents the accumulation of DNA-PKcs near the I-Sce1-induced DSB in the TK locus.
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