Repair kinetics of genomic interstrand DNA cross-links: evidence for DNA double-strand break-dependent activation of the Fanconi anemia/BRCA pathway

Abstract

The detailed mechanisms of DNA interstrand cross-link (ICL) repair and the involvement of the Fanconi anemia (FA)/BRCA pathway in this process are not known. Present models suggest that recognition and repair of ICL in human cells occur primarily during the S phase. Here we provide evidence for a refined model in which ICLs are recognized and are rapidly incised by ERCC1/XPF independent of DNA replication. However, the incised ICLs are then processed further and DNA double-strand breaks (DSB) form exclusively in the S phase. FA cells are fully proficient in the sensing and incision of ICL as well as in the subsequent formation of DSB, suggesting a role of the FA/BRCA pathway downstream in ICL repair. In fact, activation of FANCD2 occurs slowly after ICL treatment and correlates with the appearance of DSB in the S phase. In contrast, activation is rapid after ionizing radiation, indicating that the FA/BRCA pathway is specifically activated upon DSB formation. Furthermore, the formation of FANCD2 foci is restricted to a subpopulation of cells, which can be labeled by bromodeoxyuridine incorporation. We therefore conclude that the FA/BRCA pathway, while being dispensable for the early events in ICL repair, is activated in S-phase cells after DSB have formed.

FIG. 1.

Validation of the alkaline comet assay. (A) Under the conditions used in this study, control cells show a pronounced spontaneous migration of DNA (top). In contrast, ICL induced by HMT plus UVA inhibit denaturation and cause a retardation of DNA migration (bottom). (B) Dose-dependent decrease in RTM compared to that found in UVA-irradiated controls in PD743.f cells analyzed immediately after treatment with HMT plus UVA. (C) Time-dependent restoration of RTM in PD743.f cells after treatment with 0.5 ng of HMT/ml plus UVA. Complete restoration of DNA migration was observed 8 h after treatment. (D) The psoralen analog angelicin induces non-cross-linkable monoadducts. Incisions generated after treatment with 5 ng of angelicin/ml caused an increase in RTM. (E) Comparison of the incision kinetics after treatment with 0.5 ng of HMT/ml plus UVA (▪) or with 0.5 ng of HMT/ml plus UVA in the presence of 5 ng of angelicin/ml (▴) showed that parallel repair of monoadducts did not bias the detection of ICL incisions. (F) Treatment with equimolar doses of HMT plus UVA resulted in a similar initial formation of ICL in wild-type PD743.f (▪), PD720.f (FA-A) (•), and PD733.f (FA-D2) (♦) cells as well as in asynchronously growing (▪) and G₁-arrested (▴) PD743.f cells (G).

FIG. 2.

Incision kinetics of ICL in wild-type and FA cells. (A) Analysis of RTM in asynchronous PD743.f cells at different time points after treatment with HMT plus UVA showed a time-dependent restoration of DNA migration. Complete restoration of RTM (values ≥ 100%) was observed after 2 h (0.1 ng of HMT/ml plus UVA [▪]), 8 h (1 ng of HMT/ml plus UVA [▴]), and 24 h (3 ng of HMT/ml plus UVA [▾]), respectively. (B) Incision of ICL induced by 3 ng of HMT/ml plus UVA was significantly faster in G₁-arrested cells (▪) than in asynchronous cells (▴). (C) ICL were incised with similar kinetics in asynchronously growing PD720.f (FA-A [▾]), PD733.f (FA-D2 [♦]), and wild-type PD743.f (▴) cells.

FIG. 3.

DSB formation during ICL repair is dependent on DNA replication. Asynchronously growing PD743.f cells were treated with HMT+UVA and were analyzed for γH2AX staining. (A) Quantification of the RFI (see Materials and Methods) showed a dose-dependent increase in γH2AX staining starting 24 h posttreatment. At this time point, cells were arrested with a nearly 4 N DNA content (small inset). RFI values further increased at 48 h and subsequently declined. In contrast, no significant increase in RFI was observed at 4 h and 8 h posttreatment. (B) Representative images of H2AX foci in PD743.f cells 24 h after treatment with UVA alone or different concentrations of HMT plus UVA. (C) PD743.f cells were arrested in G₁ during treatment and repair (small inset) and were analyzed for γH2AX staining. No significant increases in RFI were measured throughout the time course. (D) Costaining experiments with BrDU (top panel) and γH2AX (bottom panel) confirmed that only cells positive for BrDU foci also showed γH2AX foci. In contrast, BrDU-negative cells (arrows) were also negative for γH2AX.

FIG. 4.

Initial incision is required for efficient DSB formation. (A) RMP41-77 cells (ERCC1^−/− [▴]) show clearly delayed incision kinetics compared to parental CHO cells (▪) after treatment with 1 ng of HMT/ml plus UVA. In contrast, XPG-deficient cells (▵) are able to restore DNA migration similar to that of wild-type PD743.f cells (□). (B) γH2AX formation is similarly delayed in RMP41-77 cells (light bars) compared to CHO cells (dark bars) after treatment with 0.1 (left part) and 1 ng of HMT/ml plus UVA (right part), respectively.

FIG. 5.

The FA/BRCA pathway is activated upon DSB formation in S-phase cells. (A) The kinetics of DSB formation is normal in both PD331.f (FA-C) and PD733.f (FA-D2) (B) cells. (C) Total cellular extracts of wild-type PD797.f cells were immunoblotted to detect both FANCD2 isoforms. Quantification of the fraction of Ub-FANCD2 shows a significant activation of FANCD2 only 24 h after treatment with HMT plus UVA. In contrast, activation is rapid after treatment with 10 Gy of IR. Note the clear decline in overall FANCD2 protein 24 h after IR. (D) Analysis of HeLa cells with FANCD2 foci (>10 foci per cell) confirms the rapid activation of FANCD2 after 10 Gy of IR (▪). In contrast, treatment with 1 ng of HMT/ml plus UVA (▴) does not cause activation of FANCD2-positive cells during this time (left panel). FANCD2-positve cells start to increase in a time-dependent manner only at 4 h after treatment with HMT plus UVA (right panel). (E) Costaining experiments with FANCD2 (top panel) and BrDU (bottom panel) show that only cells positive for BrDU foci also display FANCD2 foci. In contrast, BrDU-negative cells (arrows) are also negative for FANCD2 foci.

FIG. 6.

Model for the initial events of ICL repair in human cells.