Defining the Roles of Nucleotide Excision Repair and Recombination in the Repair of DNA Interstrand Cross-Links in Mammalian Cells

Abstract

The mechanisms by which DNA interstrand cross-links (ICLs) are repaired in mammalian cells are unclear. Studies in bacteria and yeasts indicate that both nucleotide excision repair (NER) and recombination are required for their removal and that double-strand breaks are produced as repair intermediates in yeast cells. The role of NER and recombination in the repair of ICLs induced by nitrogen mustard (HN2) was investigated using Chinese hamster ovary mutant cell lines. XPF and ERCC1 mutants (defective in genes required for NER and some types of recombination) and XRCC2 and XRCC3 mutants (defective in RAD51-related homologous recombination genes) were highly sensitive to HN2. Cell lines defective in other genes involved in NER (XPB, XPD, and XPG), together with a mutant defective in nonhomologous end joining (XRCC5), showed only mild sensitivity. In agreement with their extreme sensitivity, the XPF and ERCC1 mutants were defective in the incision or "unhooking" step of ICL repair. In contrast, the other mutants defective in NER activities, the XRCC2 and XRCC3 mutants, and the XRCC5 mutant all showed normal unhooking kinetics. Using pulsedfield gel electrophoresis, DNA double-strand breaks (DSBs) were found to be induced following nitrogen mustard treatment. DSB induction and repair were normal in all the NER mutants, including XPF and ERCC1. The XRCC2, XRCC3, and XRCC5 mutants also showed normal induction kinetics. The XRCC2 and XRCC3 homologous recombination mutants were, however, severely impaired in the repair of DSBs. These results define a role for XPF and ERCC1 in the excision of ICLs, but not in the recombinational components of cross-link repair. In addition, homologous recombination but not nonhomologous end joining appears to play an important role in the repair of DSBs resulting from nitrogen mustard treatment.

Arabidopsis RecQl4A suppresses homologous recombination and modulates DNA damage responses

“…Further, the response of the mutant seedlings to MMC was monitored. MMC induces DNA cross‐linking and subsequent formation of DNA double‐stranded breaks (De Silva et al. , 2000; McHugh et al.…”

“…It was intriguing that recQl4A mutant seedlings were more tolerant to MMC than the wild type. MMC is a multifunctional alkylating agent which primarily induces cross‐links between DNA strands that subsequently generate Holliday junctions during DNA replication (De Silva et al. , 2000; McHugh et al.…”

MRE11–RAD50–NBS1 is a critical regulator of FANCD2 stability and function during DNA double-strand break repair

“…DNA interstrand cross‐links (ICLs) represent a unique challenge for the repair machineries because covalent modifications that link two DNA strands preclude simple excision/repair synthesis mechanisms. Several pieces of evidence suggest that the repair of ICLs in mammalian cells occurs mainly during DNA replication, features DNA double‐strand breaks (DSBs) as repair intermediates and requires the homologous recombination machinery (Akkari et al , 2000; De Silva et al , 2000; Godthelp et al , 2002). Replication‐coupled repair of ICLs occurs when two replication forks converge at the ICL (Raschle et al , 2008).…”

SNM‐dependent recombinational repair of oxidatively induced DNA damage in Arabidopsis thaliana

“…The initial step of ICL repair involves the removal of the lesion by the NER machinery (McHugh et al , 2000). In yeast and mammals, HR also has an important role in the repair of ICLs (Jachymczyk et al , 1981; de Silva et al , 2000). Therefore, the balance between the NHEJ and HR pathways is not only critically important in the repair of DSBs but also has a strong influence on the outcome of ICL repair.…”