ASCIZ regulates lesion-specific Rad51 focus formation and apoptosis after methylating DNA damage

Abstract

Nuclear Rad51 focus formation is required for homology-directed repair of DNA double-strand breaks (DSBs), but its regulation in response to non-DSB lesions is poorly understood. Here we report a novel human SQ/TQ cluster domain-containing protein termed ASCIZ that forms Rad51-containing foci in response to base-modifying DNA methylating agents but not in response to DSB-inducing agents. ASCIZ foci seem to form prior to Rad51 recruitment, and an ASCIZ core domain can concentrate Rad51 in focus-like structures independently of DNA damage. ASCIZ depletion dramatically increases apoptosis after methylating DNA damage and impairs Rad51 focus formation in response to methylating agents but not after ionizing radiation. ASCIZ focus formation and increased apoptosis in ASCIZ-depleted cells depend on the mismatch repair protein MLH1. Interestingly, ASCIZ foci form efficiently during G1 phase, when sister chromatids are unavailable as recombination templates. We propose that ASCIZ acts as a lesion-specific focus scaffold in a Rad51-dependent pathway that resolves cytotoxic repair intermediates, most likely single-stranded DNA gaps, resulting from MLH1-dependent processing of base lesions.

Figure 1

DNA damage-induced ASCIZ focus formation. (A) Schematic diagram of ASCIZ domain organization. Circles indicate SQ/TQ motifs; ZF, Zn²⁺-finger; NLS, nuclear localization signal. (B) Time course of ASCIZ focus formation in a stable GFP-ASCIZ-expressing U2OS cell line at the indicated times after addition of 0.02% MMS. (C) Quantification of ASCIZ focus formation (green) compared to Rad51 focus formation (red). In the right panel, red bars denote cells that contain only Rad51 foci, green bars cells that contain only ASCIZ foci and yellow bars cells that contain both ASCIZ and Rad51 foci. Data are the mean of two independent experiments with >200 cells scored per sample. Similar data were obtained in other experiments. (D) Colocalization of MMS-induced ASCIZ foci with RAD51 (top) and BrdU-labeled ssDNA (bottom) in single nuclei of GFP-ASCIZ-expressing U2OS cells. (E) Flow cytometry of stable GFP-ASCIZ cells without and after 4 h treatment with 0.02% MMS, stained for DNA content with propidium iodide. (F) Immunoblots of endogenous ASCIZ in subnuclear fractions prepared from untransfected U2OS cells as described (Conlan et al, 2004), and after extraction with increasing salt buffers. Chromatin fraction indicates proteins eluted after DNase treatment of the insoluble fraction.

Figure 2

ASCIZ focus formation can occur during G1 phase. (A) Grouped data for ASCIZ focus formation in stable GFP-ASCIZ-expressing U2OS cells after 4 h treatment with 0.02% MMS in unsynchronized cells, or 4 and 12 h after release from nocodazole (noc) arrest. Asynchronous cells were incubated with BrdU during MMS treatment; synchronized cells were released into BrdU-containing medium before MMS treatment. Data are the mean±s.e. of three independent experiments, scoring >250 cells per sample. (B) Kinetics of S-phase entry, detected by BrdU incorporation, in the same cell line after release from nocodazole arrest. Propidium iodide-stained FACS profiles of a similar experiment are shown in Supplementary Figure S5. (C) Examples of micrographs of MMS-treated cells at 4 or 12 h after nocodazole release (top panels), and grouped data of the fractional distribution of BrdU and ASCIZ foci indices, scored as in (A). Arrows indicate examples of G1 cells containing ASCIZ foci and arrowheads indicate post-G1 cells containing ASCIZ foci.

Figure 3

ASCIZ is required for Rad51 focus formation and cell survival in response to MMS treatment. (A) Western blot analysis of ASCIZ, Rad51 and actin from ASCIZ siRNA- and luciferase siRNA-treated U2OS cells. (B) Apoptosis in U2OS cells siRNA-treated as in (A) at the indicated times after 0.005% MMS addition (mean±s.e. of three independent experiments), detected by TUNEL staining. ^*P<0.01 (paired t-test, two-sided) versus mock and GL2 at 24 h. (C) MMS-induced Rad51 focus formation in siRNA-treated U2OS cells. The bottom panel shows enlargements of nuclei labeled by arrows above. (D) Quantitation of Rad51 focus formation (mean±s.e. of 2–3 independent experiments) in siRNA-treated U2OS cells under basal conditions and in response to MMS or IR as in (B) and (E). Similar results for another ASCIZ siRNA are shown in Supplementary Figure S6C. (E) γH2AX formation in siRNA-treated U2OS cells with or without MMS treatment. The right panel shows an overexposed Western blot demonstrating the specificity of the antibody. In the experiment shown here, 0.14 and 99.5% of control and 0.25 and 97.1% of ASCIZ-depleted cells contained γH2AX in the absence or presence of MMS treatment, respectively (>170 nuclei scored per sample). (F) Formation of Rad51 foci but not GFP-ASCIZ foci in response to IR (left panels), and normal Rad51 focus formation after IR in ASCIZ siRNA-treated U2OS cells (right panels).

Figure 4

ASCIZ siRNA phenotypes and expression profiles. (A) Flow cytometry of luciferase siRNA- and ASCIZ siRNA-treated U2OS cells, stained with propidium iodide. Cell cycle distribution was analyzed using ModFit LT. (B) S-phase indices of siRNA-treated U2OS cells pulse-labeled for 40 min with 10 μg/ml BrdU. (C) Colony formation of siRNA-treated U2OS cells. (D, E) Northern blot analysis of ASCIZ and GAPDH mRNAs in normal human tissues (D) and human cancer cell lines (E) as indicated. For each cell line, samples were prepared from semiconfluent (left lanes) and confluent (right lanes) cultures. Mass standards (kb) are indicated on the left. The mRNAs correspond to two differently sized ASCIZ cDNAs in GenBank (BC002701 and NM_015251). (F) Western blot analysis of endogenous ASCIZ in cytosol (C) and nuclear (N) fractions of the cell lines indicated above. The position of a 97 kDa marker band is indicated on the right, and actin levels are shown below as a loading control.

Figure 5

Regulation of ASCIZ focus formation and DNA damage-induced apoptosis. (A) Dose response of ASCIZ focus formation in the stable GFP-ASCIZ-expressing U2OS cell line after 6 h MNNG treatment. The micrograph was taken 6 h after addition of 20 μM MNNG. (B) Dose response of ASCIZ focus formation in the same cell line after 4 h MMS treatment in the presence (filled bars) or absence (hatched bars) of 6 mM methoxyamine (MOA). (C) ASCIZ focus formation in transiently transfected HCT116 cells (open bars), or HCT116 cells complemented with chromosome 2 or 3 without or after 4 h 0.02% MMS treatment. ^*P<0.01% versus all other samples (paired t-test, two-sided). (D) Apoptosis in the HCT116 cell lines treated with luciferase (LUC) or ASCIZ (ASC) siRNA 24 h after addition of 0.005% MMS. The inset shows an anti-ASCIZ Western blot of the chromosome 2 and 3 complemented HCT116 lines treated with luciferase (L) or ASCIZ (A) siRNAs, using crossreacting bands on the same blot as a loading control. (E) ASCIZ focus formation in stable GFP-ASCIZ-expressing U2OS cells in response to the indicated H₂O₂ doses (mean±s.e. of 3–4 independent experiments; >200 cells per sample scored).

Figure 6

Identification of a focus-forming ASCIZ core domain. (A) Schematic diagram of ASCIZ domains with arrows indicating truncation points. (B, C) Representative micrographs of focus formation by the indicated ASCIZ fragments in the absence or presence of 0.02% MMS for 4 h in transiently transfected U2OS cells, and co-staining of GFP-ASCIZ residues 67–286 with Rad51 (C, left) and PML antibodies (C, right). Note that micrographs were taken from densely grown fields of cells, and that Rad51 foci are considerably more intense in ASCIZ core domain-expressing cells.

Figure 7

Summary model. Primary methylating or oxidative base damage is converted to abasic sites, which are further processed into ssDNA gaps in an MLH1-dependent manner. ssDNA gaps lead to the formation of ASCIZ foci that recruit Rad51 for DNA repair to prevent DNA damage-induced apoptosis.