End-joining of blunt DNA double-strand breaks in mammalian fibroblasts is precise and requires DNA-PK and XRCC4
D N A Repair , Volume 3 - Issue 1 p. 43- 50
DNA double-strand break repair by non-homologous end-joining (NHEJ) is generally considered to be an imprecise repair pathway. In order to study repair of a blunt, 5′ phosphorylated break in the DNA of mammalian fibroblasts, we used the E. coli cut-and-paste type transposon Tn5. We found that the Tn5 transposase can mediate transposon excision in Chinese hamster cell lines. Interestingly, a blunt 5′ phosphorylated break could efficiently be repaired without loss of nucleotides in wild type fibroblasts. Catalytic subunit of DNA-dependent protein kinase (DNA-PKCS) deficiency reduced the efficiency of joining four-fold without reducing precision, whereas both efficiency and accuracy of joining were affected in Ku80 or XRCC4 mutant cell lines. These results show that both the DNA-PK and the XRCC4/ligase IV complexes are required for NHEJ and that other, more error-prone, repair processes cannot efficiently substitute for joining of blunt breaks produced in living cells. Interestingly, the severity of the end-joining defect differs between the various mutants, which may explain the difference in the severity of the phenotypes, which have been observed in the corresponding mouse models.
|DNA repair, Tn5 transposase, Transposition, V(D)J recombination|
|D N A Repair|
|Organisation||Department of Molecular Genetics|
van Heemst, D, Brugmans, L.J.L, Verkaik, N.S, & van Gent, D.C. (2004). End-joining of blunt DNA double-strand breaks in mammalian fibroblasts is precise and requires DNA-PK and XRCC4. D N A Repair, 3(1), 43–50. doi:10.1016/j.dnarep.2003.09.004