Abstract
Chromosomal double strand breaks (DSBs) occurring in human genome are generally repaired either of two pathways; End-joing (EJ) or homologous recombination (HR). We recently developed a system to trace the fate of DSBs occurring in human genome using meganuclease I-SceI site (Honma et al., EMM 42, 288-298, 2003). The lymphoblastoid cell lines TSCE5 and TSCE105 have single and double I-SceI site in the thymidine kinase gene (TK), respectively. Introducing highly efficient DSBs into the cells using NucleofectorTM (Amaxa) made it possible to recover mutants by the DSBs without the phenotypic TK selection. The mutant frequencies of TSCE5 and TSCE105 were 3% and 30%, respectively. Most of mutations in TSCE5 were small deletions ranging from 1 to 30bp and others were over 30bp deletions and DNA rearrangements. On the other hand, TSCE105 mainly produced large deletions encompasing the two I-SceI sites. No mutants resulted by HR was observed in both cells. These results support an idea that majority of DSBs are repaired by EJ resulting small deletion, and HR rarely contributes for that. Error-free EJ mechanism may be possible, because single DSB in TSCE5 did not efficiently cause deletional mutations than expected.
