An ATR- and BRCA1-mediated Fanconi anemia pathway is required for activating the G2/M checkpoint and DNA damage repair upon rereplication

Abstract

The timely assembly of prereplicative complexes at replication origins is tightly controlled to ensure that genomic DNA is replicated once per cell cycle. The loss of geminin, a DNA replication inhibitor, causes rereplication that activates a G2/M checkpoint in human cancer cells. Fanconi anemia (FA) is an autosomal recessive and X-linked disorder associated with cancer susceptibility. Here we show that rereplication activates the FA pathway both for the activation of a G2/M checkpoint and for repair processes, like recruitment of RAD51. Both ATR and BRCA1 are required to activate the FA pathway. The G2/M checkpoint-mediated arrest of the cell cycle is critical for the prevention of both apoptosis and the accumulation of cells with rereplicated DNA, because the loss of ATR, BRCA1, or FANCA promotes apoptosis and suppresses the accumulation. The accumulation of cells with rereplicated DNA is restored by the artificial induction of a G2-phase arrest even when ATR, BRCA1, or FANCA is absent. Therefore, the ATR- and BRCA1-mediated FA pathway is required for the activation of a G2/M checkpoint and for DNA damage repair in response to the endogenous signal of rereplication. In its absence, the cells rapidly lose viability when faced with rereplication.

FIG. 1.

Depletion of geminin leads to generation of both ssDNA and double-stranded breaks in human cancer cells. (A) Double-stranded breaks are generated in geminin-depleted cells. HCT116 cells were transfected with control siRNA oligonucleotide GL2 or geminin siRNA oligonucleotide GEM twice and harvested 72 h after the first transfection. Shown are results from the Comet assay done under neutral conditions, which detects primarily double-stranded DNA breaks, and alkaline unwinding conditions, which detects both double-stranded and single-stranded DNA breaks. (B) Percentages of cells with DNA damage under both neutral and alkaline unwinding conditions as shown in panel A. At least 80 cells were counted for each experiment. Means ± standard deviations from two experiments are shown. (C) ssDNA is generated in geminin-depleted cells. HCT116 cells were transfected with control siRNA oligonucleotide GL2 or geminin siRNA oligonucleotide GEM with the addition of BrdU after the first transfection and harvested 72 h after the first transfection. Coimmunostaining with anti-BrdU antibody without denaturation and phospho-H2AX antibody (P-H2AX) (Ser139) is shown.

FIG. 2.

FA pathway is activated in geminin-depleted cells. (A) Association of RPA70 and FANCD2 with chromatin. HCT116 cells were transfected with control siRNA oligonucleotide GL2 or geminin siRNA oligonucleotide GEM as described in the legend for Fig. 1A. Chromatin fractionation was performed as described by Zou et al. (51). The resultant fractions were resolved on sodium dodecyl sulfate-polyacrylamide gels and immunoblotted with the indicated antibodies (FANCD2-L is monoubiquitinated, and FANCD2-S is unubiquitinated). Orc2 served as a loading control for the chromatin fractions (Chr.) *, protein cross-reacting with anti-ORC2 antibody served as a loading control for the soluble fractions (Sol.). (B) FANCD2 colocalizes with single-stranded BrdU in geminin-depleted cells. HCT116 cells treated as described in the legend for Fig. 1A were immunostained with anti-FANCD2 and anti-BrdU antibodies without denaturation.

FIG. 3.

FA proteins are required for rereplication and activation of the G₂/M checkpoint in geminin-depleted cells. (A) Schematic of transfection protocol. (B) HCT116 cells treated as shown in panel A were fractionated as described in the legend for Fig. 2A. The resultant fractions were resolved on sodium dodecyl sulfate-polyacrylamide gels and immunoblotted with the indicated antibodies. See the legend for Fig. 2A for additional explanations. (C) HCT116 cells treated as shown in panel A were harvested for flow cytometry analysis. Cells with more than G₂/M DNA content are regarded as cells with rereplicated DNA. Primary data are shown in Fig. S1 in the supplemental material. (D) HCT116 cells treated as shown in panel A were immunoblotted for the indicated proteins. (E) Cell cycle distribution of transfected cells. HCT116 cells treated as shown in panel A or Fig. 5A were stained with propidium iodide and subjected to FACS analysis. >G2, cells with more than G₂/M DNA content (Cellquest).

FIG. 4.

FANCD2 is required for the accumulation of Rad51 foci in cells undergoing rereplication. (A) HCT116 cells transfected as shown in Fig. 3A were coimmunostained with anti-Rad51 and anti-FANCD2 antibodies. (B) Rad51 focus-positive cells after different siRNA transfections. Cells containing more than five Rad51 foci in the nucleus were scored as focus-positive cells. For GEM or GEM plus FANCD2 siRNA-transfected cells, only cells with giant nuclei were counted. At least 100 cells were examined and the percentages of positive cells calculated from two independent experiments.

FIG. 5.

ATR and BRCA1 are required for FA protein-mediated G₂/M checkpoint activation. (A) Schematic of transfection protocol. (B) HCT116 cells treated as shown in panel A were harvested for flow cytometry analysis. Cells with more than G₂/M DNA content are regarded as cells with rereplicated DNA. (C) HCT116 cells treated as shown in panel A were immunoblotted for the indicated proteins. (D) HCT116 cells were treated as shown in panel A. The resultant fractions were resolved on sodium dodecyl sulfate-polyacrylamide gels and immunoblotted with the indicated antibodies. See the legend for Fig. 2A for additional explanations. (E) HCC1937 (BRCA1^+/+) and HCC1937 (BRCA1^−/−) cells were transfected with control oligonucleotide GL2 or GEM twice and harvested 72 h after the first transfection for flow cytometry analysis (left panel) or immunoblotting with the indicated antibodies (right panel). *, protein cross-reacting with antigeminin antibody served as a loading control. FL2, filter that detects fluorescence with emission range at 595 ± 20 nm.

FIG. 6.

Premitotic arrest bypasses the requirement of a checkpoint pathway for cells to accumulate rereplicated DNA. (A) Schematic of transfection protocol. (B) Silencing cyclin A rescues the accumulation of cells with overreplicated DNA in geminin and ATR or geminin and BRCA1 cosilenced cells. HCT116 cells treated as shown in panel A were harvested for flow cytometry analysis. (C) HCT116 cells treated as shown in panel A were immunoblotted with the indicated antibodies. (D) Schematic of transfection protocol. (E) Silencing cyclin A rescues the accumulation of cells with overreplicated DNA in geminin and FANCA cosilenced cells. HCT116 cells treated as shown in panel D were harvested for flow cytometry analysis.

FIG. 7.

Downregulation of geminin in ATR-, BRCA1-, FANCA-, or FANCD2-depleted cells causes apoptosis. (A) HCT116 cells were codepleted of geminin and ATR (Fig. 5A), BRCA1 (Fig. 5A), FANCA (Fig. 3A), or FANCD2 (Fig. 3A) and harvested 72 h after the first transfection for immunoblotting using the indicated antibodies. (B) HCT116 cells were treated as shown in Fig. 3A or 5A, and cell viability was measured by an MTT cell growth assay kit (Promega) according to the manufacturer's instructions. OD, optical density.

FIG. 8.

Rereplication-induced activation of the FA pathway is required for activation of the G₂/M checkpoint and DNA damage repair. Rereplication by loss of geminin generates double-stranded breaks (DSB) and ssDNA. The ssDNA is sensed through ATR and BRCA1, resulting in activation of the FA pathway. The pathway bifurcates at the Fanconi core complex to activate the G₂/M checkpoint and to monoubiquitinate FANCD2. The DNA damage repair initiated by active FANCD2 appears essential to prevent apoptosis and short-term loss of viability.