Abstract
DNA interstrand cross-links (ICLs) are the DNA damage that both strand of the double helix are covalently joined by single ICL agents widely used as a potent antitumor drugs. Since, ICLs prevents strand separation, DNA replication, transcription and recombination are strongly blocked by this lesions. Despite the medical relevance of ICL, detail of its repair in mammalian cells remain unclear. Here, we have developed the psoralen-based quantitative assay system (Psoralen-PEO-biotin excision assay: PPBE assay), which can detect 10 - 20 ICLs in a mammalian cell. This method can detect the complete removal of the ICL agents from the genome DNA. The analysis using this system revealed that FA-G and FA-A cells are deficient in removal of ICLs, while repair proficient cells can remove up to 2,500 ICLs in 48 hrs. Furthermore, normal kinetics of removal was observed in homologous recombination deficient cells, but not in XPF, MSH2 and Rev3 deficient cells. These results indicated that FANC core complex proteins, XPF, MSH2 and Rev3 are required for removal of ICL, possibly at an earlier step than homologous recombination in ICL repair.
