No abstract

In response to DNA damage or replication fork stress, the Fanconi anemia (FA) pathway is activated, leading to monoubiquitination of FancD2 and FancI and their co-localization in foci. Here we show that, in the chicken DT40 cell system, multiple alanine-substitution mutations in 6 conserved and clustered S/TQ motifs of FancI largely abrogate monoubiquitination as well as focus formation of both FancI and FancD2, resulting in loss of DNA repair function. Conversely, FancI carrying phospho-mimic mutations on the same 6 residues induces constitutive monoubiquitination and focus formation of FancI and FancD2, and protects against cell killing and chromosome breakage by DNA interstrand crosslinking agents. We propose that the multiple phosphorylation of FancI serves as a molecular switch in activation of the FA pathway. Mutational analysis of putative phosphorylation sites in human FANCI indicates that this switch is evolutionarily conserved.

Parp-1 and Parp-2 are activated by DNA breaks and have been implicated in the repair of DNA single-strand breaks (SSB). Their involvement in double-strand break (DSB) repair mediated by homologous recombination (HR) or nonhomologous end joining (NHEJ) remains unclear. We addressed this question using chicken DT40 cells, which have the advantage of carrying only a PARP-1 gene but not a PARP-2 gene. We found that PARP-1 À/À DT40 mutants show reduced levels of HR and are sensitive to various DSB-inducing genotoxic agents. Surprisingly, this phenotype was strictly dependent on the presence of Ku, a DSBbinding factor that mediates NHEJ. PARP-1/KU70 double mutants were proficient in the execution of HR and displayed elevated resistance to DSB-inducing drugs. Moreover, we found deletion of Ligase IV, another NHEJ gene, suppressed the camptothecin of PARP-1 À/À cells. Our results suggest a new critical function for Parp in minimizing the suppressive effects of Ku and the NHEJ pathway on HR.

Cross-linking agents that induce DNA interstrand cross-links (ICL) are widely used in anticancer chemotherapy. Yeast genetic studies show that nucleotide excision repair (NER), Rad6/Rad18-dependent postreplication repair, homologous recombination, and cell cycle checkpoint pathway are involved in ICL repair. To study the contribution of DNA damage response pathways in tolerance to cross-linking agents in vertebrates, we made a panel of gene-disrupted clones from chicken DT40 cells, each defective in a particular DNA repair or checkpoint pathway, and measured the sensitivities to cross-linking agents, including cis-diamminedichloroplatinum (II) (cisplatin), mitomycin C, and melphalan. We found that cells harboring defects in translesion DNA synthesis (TLS), Fanconi anemia complementation groups (FANC), or homologous recombination displayed marked hypersensitivity to all the cross-linking agents, whereas NER seemed to play only a minor role. This effect of replicationdependent repair pathways is distinctively different from the situation in yeast, where NER seems to play a major role in dealing with ICL. Cells deficient in Rev3, the catalytic subunit of TLS polymerase PolZ, showed the highest sensitivity to cisplatin followed by fanc-c. Furthermore, epistasis analysis revealed that these two mutants work in the same pathway. Our genetic comprehensive study reveals a critical role for DNA repair pathways that release DNA replication block at ICLs in cellular tolerance to cross-linking agents and could be directly exploited in designing an effective chemotherapy. (Cancer Res 2005; 65(24): 11704-11)