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.DNA interstrand cross-links (ICL) are among the most cytotoxic of all DNA lesions, and the presence of a single unrepaired ICL is sufficient to kill repair-deficient bacteria and yeast (27,29). ICL effectively prevent the separation of DNA strands and therefore block essential cellular processes, e.g., transcription and DNA replication and recombination. DNA ICL agents such as mitomycin C, cisplatin and photoactivated psoralens are widely used as potent antitumor drugs (33). The importance of understanding details of ICL repair pathways is also highlighted by the hypersensitivity to ICL-inducing agents in the human genetic disease Fanconi anemia (FA) and in cells mutant in the breast cancer genes 46,58).Despite the medical relevance of this class of DNA damage, details of ICL repair in human cells remain largely unknown. Because both strands of the DNA are simultaneously affected and because no undamaged strand is available as template, ICL repair is thought to be complex and appears to involve several different pathways (for a recent review see reference 15). We have previously shown that psoralen-treated primary fibroblasts arrest in the late S phase regardless of when the damage was introduced during the cell cycle (2). FA mutant cells displayed a significantly prolonged S-phase arrest, suggesting that the FA pathway acts in an S-phase-specific damage response during ICL repair (1). This hypothesis was strengthened by the recent discovery that BRCA2, known to control homologous recombination (HR) of DNA double-strand breaks (DSB) (26,35), is mutated in the FA complementation groups B and D1 (21). Accordingly, a model for ICL repair in human cells has been proposed, in which repair events are restricted to the S phase (30). The model predicts that, during DNA ...

The comet assay (single-cell gel electrophoresis) is a simple and sensitive method for studying DNA damage and repair. In this microgel electrophoresis technique, a small number of cells suspended in a thin agarose gel on a microscope slide is lysed, electrophoresed, and stained with a fluorescent DNA-binding dye. Cells with increased DNA damage display increased migration of chromosomal DNA from the nucleus towards the anode, which resembles the shape of a comet. The assay has manifold applications in fundamental research for DNA damage and repair, in genotoxicity testing of novel chemicals and pharmaceuticals, environmental biomonitoring, and human population monitoring. This chapter describes a standard protocol of the alkaline comet assay and points to some useful modifications.

No abstract

SUMMARY Accumulation of toxic metabolites in tyrosinemia type I (HT1) patients leads to chronic DNA damage and the highest risk for hepatocellular carcinomas (HCCs) of any human disease. Here we show that hepatocytes of HT1 mice exhibit a profound cell cycle arrest which, despite concomitant apoptosis resistance, causes mortality from impaired liver regeneration. However, additional loss of p21 in HT1 mice restores the proliferative capabilities of hepatocytes and renal proximal tubular cells. This growth response compensates cell loss due to uninhibited apoptosis and enables animal survival but rapidly leads to HCCs, renal cysts and renal carcinomas. Thus, p21’s antiproliferative function is indispensable for the suppression of carcinogenesis from chronically injured liver and renal epithelial cells and cannot be compensated by apoptosis.