End-joining of blunt DNA double-strand breaks in mammalian fibroblasts is precise and requires DNA-PK and XRCC4.
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-PK(CS)) 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.
van Heemst
Diana
D
Department of Cell Biology and Genetics, Erasmus Medical Center, P.O. Box 1738, 3000 DR, Rotterdam, The Netherlands. d.vangent@erasmusmc.nl
Brugmans
Linda
L
Verkaik
Nicole S
NS
van Gent
Dik C
DC
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D004251:DNA Transposable Elements; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*
D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; C101467:XRCC4 protein, human; D004247:DNA; D051747:DNA-Activated Protein Kinase; D017346:Protein Serine-Threonine Kinases