DNA ligase I deficiency leads to replication-dependent DNA damage and impacts cell morphology without blocking cell cycle progression

Abstract

46BR.1G1 cells derive from a patient with a genetic syndrome characterized by drastically reduced replicative DNA ligase I (LigI) activity and delayed joining of Okazaki fragments. Here we show that the replication defect in 46BR.1G1 cells results in the accumulation of both single-stranded and double-stranded DNA breaks. This is accompanied by phosphorylation of the H2AX histone variant and the formation of gammaH2AX foci that mark damaged DNA. Single-cell analysis demonstrates that the number of gammaH2AX foci in LigI-defective cells fluctuates during the cell cycle: they form in S phase, persist in mitosis, and eventually diminish in G(1) phase. Notably, replication-dependent DNA damage in 46BR.1G1 cells only moderately delays cell cycle progression and does not activate the S-phase-specific ATR/Chk1 checkpoint pathway that also monitors the execution of mitosis. In contrast, the ATM/Chk2 pathway is activated. The phenotype of 46BR.1G1 cells is efficiently corrected by the wild-type LigI but is worsened by a LigI mutant that mimics the hyperphosphorylated enzyme in M phase. Notably, the expression of the phosphomimetic mutant drastically affects cell morphology and the organization of the cytoskeleton, unveiling an unexpected link between endogenous DNA damage and the structural organization of the cell.

FIG. 1.

DNA breaks accumulate in LigI-defective cells. (A) Total cell extracts of the indicated cell lines were analyzed by Western blotting with anti-γH2AX and anti-α-tubulin antibodies. Extracts in the right panels were prepared from 46BR.1G1 cells before (−) or after (+) treatment with etoposide. (B) Representative images of γH2AX foci. 7A3, 46BR.1G1, and M10 cells were analyzed in indirect immunofluorescence assays with an anti-γH2AX antibody and counterstained with DAPI. Bar, 10 μm. (C and D) Alkaline (C) and neutral (D) comet assay analysis of 7A3, 46BR.1G1, and M10 cells. Distribution of the tail moment of comets in 7A3 (light gray bars), 46BR.1G1 (medium gray bars), and M10 (dark gray bars) cells. Data are calculated by the analysis of 450 to 550 cells for each cell line in four experiments. Results show means ± standard errors of the means. Cells are grouped in seven categories based on the value of comet tail moment.

FIG. 2.

Replication intermediates accumulate in LigI-defective cell lines. BrdU comet analysis of 7A3, 46BR.1G1, and M10 cells. Cells were pulse-labeled with BrdU for 15 min and either immediately processed or chased for 1, 5, or 10 h prior to comet processing. (A) Representative images of 7A3 and M10 cells. Identical fields are shown with green fluorescein (BrdU) and blue DAPI (DNA) staining. (a and g) BrdU staining after the pulse; (d and l) 1-h chase; (b, h, e, and m) the same cells stained with DAPI; (c, f, i, and n) merged images. (B) Quantification of the BrdU comet assay at the indicated chase periods. Results are based on the analysis of 50 to 60 comets/point in four experiments. Bars show means ± standard errors of the means.

FIG. 3.

Analysis of replication intermediates. 7A3 and M10 cells were pulse-labeled for 15 min with [³H]thymidine. Purified DNA was heat denatured and fractionated through 5 to 30% neutral sucrose gradients. Fractions were taken from the top of the gradient. Aliquots of each fraction were counted to determine the distribution of nascent DNA in 7A3 and M10 cells. Arrows, DNA size markers in kilobases. The top of the gradient is on the left.

FIG. 4.

Chronic activation of DNA damage signaling in LigI-defective cells. (A) Western blotting of MRC-5V1, 46BR.1G1, 7A3, and M10 total cell extracts with antibodies against the indicated proteins or phosphoepitopes. The same analysis was performed in 46BR.1G1 cells after etoposide treatment (+ et). (B) Asynchronous MRC-5V1, 7A3, 46BR.1G1, and M10 cells were costained with anti-γH2AX and anti-cyclin A antibodies. γH2AX-positive cells were scored as having more than five distinct foci in the nucleus. For each cell line, the fraction of nuclei displaying γH2AX foci was determined in 100 randomly selected cells in three experiments. Bars show means ± standard errors of the means. (C) Quantitation of γH2AX-positive cells in G₁ phase (cyclin A negative) and in S/G₂ phases (cyclin A positive) in MRC-5V1, 7A3, 46BR.1G1, and M10 cells. Bars show means ± standard errors of the means.

FIG. 5.

LigI-deficient cells enter a new cell cycle with signs of unrepaired DNA breaks. (A and B) Asynchronous M10 cells were costained with anti-γH2AX and anti-phospho-histone H3 (H3-p) antibodies. DNA was stained with DAPI. Confocal images. Bars, 10 μm. Panel B shows a cell in telophase. (C) Quantitation of γH2AX foci in mitotic MRC-5V1, 7A3, 46BR.1G1, and M10 cells. Cells were stained as in panel A, and mitotic cells were scored for the number of γH2AX foci. About 100 mitotic cells were scored for each cell line. Results are pooled from two independent experiments. (D) Cells were pulse-labeled with BrdU for 15 min and either immediately processed (pulse) or chased for 10 h prior to fixation (chase). Representative images of M10 cells, where identical fields are shown with green fluorescein staining (BrdU) and red TRITC staining (cyclin A). Nuclei were stained with DAPI. Arrowheads, BrdU-positive nuclei. Bar, 10 μm. (E) 7A3, 46BR.1G1, and M10 cells were pulse-labeled with BrdU as described for panel D and scored for cyclin A-negative nuclei (G₁) with mid/late patterns of BrdU replication foci. Frequency distribution plots of cyclin A-negative cells with mid/late replication foci after a chase period of 10 h. Results show means ± standard errors of the means of three experiments.

FIG. 6.

The expression of LigI-4D mutant affects cell morphology. Morphology of 46BR.1G1, 7A3, and M10 cells. (A) Cells were grown on coverslips, and images were taken by phase-contrast microscopy at subconfluent cell density. (B) Cells were decorated with TRITC-conjugated phalloidin. Nuclei were counterstained with DAPI. Arrows show bundles of actin filaments (stress fibers). (C) Cells were decorated with antivinculin antibody and TRITC-conjugated anti-mouse secondary antibody. Nuclei were counterstained with DAPI. Arrowheads show focal contacts. Bars = 10 μm.