DNA-PK-independent rejoining of DNA double-strand breaks in human cell extracts in vitro

Purpose: To investigate the role of DNA-dependent protein kinase (DNA-PK) i n the rejoining of ionizing radiation-induced DNA double-strand breaks (dsb ). Materials and methods: This study employed previously described in vitro as says that utilize nuclei or 'naked' DNA prepared from agarose-embedded cell s as a substrate and S-HeLa cell extracts as a source of enzymes. Rejoining of dsb in these assays is absolutely dependent on cell extract and it proc eeds, under optimal reaction conditions, to an extent similar to that obser ved in intact cells. Results were confirmed in a plasmid-based assay for in vitro rejoining of dsb. Results: It is shown that concentrations of wortmannin completely inhibitin g DNA-PK activity profoundly affect the rejoining df dsb in vivo, but have no effect on dsb rejoining in vitro. Furthermore, fractionation of cell ext racts using ammonium sulphate precipitation, generates protein fractions th at are able to support dsb rejoining, despite the fact that they do not con tain detectable amounts of either DNA-PKcs or Ku80. Efficient rejoining of dsb in vitro is also observed with extracts of MO59J cells that lack DNA-PK activity. Finally, rejoining of dsb remains unaffected by wortmannin in a plasmid-based assay, and is also detectable with extracts of MO59J cells. Conclusions: These findings are in contrast with genetic studies demonstrat ing a requirement for DNA-PK activity for efficient rejoining of dsb in viv o. The difference between in vitro and in vivo results may not be attribute d to chromatin structure since wortmannin was without an effect when using nuclei as a substrate. It is speculated that the differences between in viv o and in vitro results can be explained either by assuming the operation of multiple pathways in dsb rejoining, some of which do not require DNA-PK, o r by postulating a purely regulatory/damage sensing role for DNA-PK in inta ct cells but no direct involvement in dsb rejoining.