In mammalian cells, ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) are repaired in all phases of the cell cycle predominantly by classical, DNA-PK-dependent nonhomologous end joining (D-NHEJ). Homologous recombination repair (HRR) is functional during the S- and G2-phases, when a sister chromatid becomes available. An error-prone, alternative form of end joining, operating as backup (B-NHEJ) functions robustly throughout the cell cycle and particularly in the G2-phase and is thought to backup predominantly D-NHEJ. Parp-1, DNA-ligases 1 (Lig1) and 3 (Lig3), and Xrcc1 are implicated in B-NHEJ. Chromosome and chromatid translocations are manifestations of erroneous DSB repair and are crucial culprits in malignant transformation and IR-induced cell lethality. We analyzed shifts in translocation formation deriving from defects in D-NHEJ or HRR in cells irradiated in the G2-phase and identify B-NHEJ as the main DSB repair pathway backing up both of these defects at the cost of a large increase in translocation formation. Our results identify Parp-1 and Lig1 and 3 as factors involved in translocation formation and show that Xrcc1 reinforces the function of Lig3 in the process without being required for it. Finally, we demonstrate intriguing connections between B-NHEJ and DNA end resection in translocation formation and show that, as for D-NHEJ and HRR, the function of B-NHEJ facilitates the recovery from the G2-checkpoint. These observations advance our understanding of chromosome aberration formation and have implications for the mechanism of action of Parp inhibitors.

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Nucleoside analogs (NSAs) were among the first chemotherapeutic agents and could also be useful for the manipulation of cell fate. To investigate the potential of NSAs for the induction of neuronal differentiation, we developed a novel phenotypic assay based on a human neuron-committed teratocarcinoma cell line (NT2) as a model for neuronal progenitors and constructed a NT2-based reporter cell line that expressed eGFP under the control of a neuron-specific promoter. We tested 38 structurally related NSAs and determined their activity to induce neuronal differentiation by immunocytochemistry of neuronal marker proteins, live cell imaging, fluorometric detection and immunoblot analysis. We identified twelve NSAs, which induced neuronal differentiation to different extents. NSAs with highest activity carried a halogen substituent at their pyrimidine nucleobase and an unmodified or 2'-O-methyl substituted 2-deoxy-β-D-ribofuranosyl residue as glyconic moiety. Cladribine, a purine nucleoside with similar structural features and in use to treat leukemia and multiple sclerosis, induced also differentiation of adult human neural crest-derived stem cells. Our results suggest that NSAs could be useful for the manipulation of neuronal cell fate in cell replacement therapy or treatment of neurodegenerative disorders. The data on the structure and function relationship will help to design compounds with increased activity and low toxicity.

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