Impairment of homologous recombination control in a Fanconi anemia‐like Chinese Hamster Cell mutant

Abstract

DNA interstrand cross-links (ICL)-inducing agents such as cisplatin, mitomycin C (MMC) and nitrogen mustards are widely used as potent antitumor drugs. Although ICL repair mechanism is not yet well characterized in mammalian cells, this pathway is thought to involve a sequential action of nucleotide excision repair (NER) and homologous recombination (HR). The importance of unraveling ICL repair pathways is highlighted by the hypersensitivity to ICL-inducing agents in cells of patients with the genetic disease Fanconi anemia (FA) and in cells mutated in the Breast Cancer susceptibility genes BRCA1 and BRCA2. To better characterize the involvement of HR in the sensitivity to ICL-inducing agents, we examined spontaneous and ICL-induced HR in rodent FA-like V-H4 cells. In this report, we show that MMC-hypersensitive V-H4 cells exhibit an increased spontaneous homology-directed repair (HDR) activity compared to the resistant V79 parental cells. Elevated HDR activity results mainly in increased conservative Rad51-dependent recombination, without affecting nonconservative single-strand annealing process (SSA). We also show that HDR activity is enhanced following MMC treatment in parental cells, but not in rodent FA-like V-H4 cells. Moreover, our data indicate that Rad51 foci formation is significantly delayed in these FA-like cells in response to crosslinking agent. These findings provide evidence for an impairment of HR control in V-H4 cells and emphasize the involvement of the FA pathway in HR-mediated repair.

Hypersensitivity of Brca1-deficient MEF to the DNA interstrand crosslinking agent mitomycin C is associated with defect in homologous recombination repair and aberrant S-phase arrest

“…Brca1 is colocalized with Rad51 to form IR-induced foci (Scully et al, 1997;Chen et al, 1999b). Consistently, Rad51 foci formation is delayed in the FA-like cell in response to ICL agents (Larminat et al, 2004), and Brca1-null ES cell shows a reduced level of Rad51 foci after cisplatin treatment (Bhattacharyya et al, 2000). Our results also show that Rad51 foci formation following MMC treatment is significantly impaired in Brca1-deficient MEFs but is restored by reconstitution of Brca1 expression ( Figure 5).…”

Disruption of Murine Mus81 Increases Genomic Instability and DNA Damage Sensitivity but Does Not Promote Tumorigenesis

“…Focus formation is dependent on a number of upstream activities, and the foci are thought to represent sites of homologous recombination (26,35,37). A number of mutant cell lines that are extremely sensitive to MMC show delayed or defective Rad51 focus formation (6,34). A defect in Rad51 focus formation in Mus81-deficient cells would suggest that Mus81 functions upstream of the strand invasion step in the homologous repair pathway.…”

p21CDKN1A allows the repair of replication-mediated DNA double-strand breaks induced by topoisomerase I and is inactivated by the checkpoint kinase inhibitor 7-hydroxystaurosporine

“…Abbreviations: CL ¼ control untreated cells; R0 ¼ at the end of the treatment with 1 mM of camptothecin for 3 h; R6 ¼ 6 h after removal of camptothecin. Hoeijmakers, 2001;Larminat et al, 2004). After DNA damage, Rad51 is overexpressed and forms foci associated with p21 CDKN1A expression (Raderschall et al, 2002).…”