Heat sensitivity of double-stranded DNA-dependent protein kinase (DNA-PK) activity

Purpose: The heat sensitivity of DNA-PK activity in hybrid cells and the possible restoration of this activity with extracts from scid cells (defective in DNA-PKcs), sxi-3 cells (defective in Ku80) and V79-4 (sxi-3 parental wild-type cells) was analysed. Materials and methods: Heat treatment of cells was performed in a water bath at 44 degrees C. The cell extract from scid cells or sxi-3 cells was added to heat-treated hybrid cell extracts, and the DNA-PK activity was assayed. Results: When hybrid cells were heated at 44 degrees C for 15 min, DNA-PK activity was reduced to undetectable levels. The decreased DNA-PK activity could be restored in a concentration-dependent manner with the addition of scid cell extract. The sxi-3 cell extract could not restore heat-inactivated DNA-PK activity. Conclusions: DNA-PK was inactivated by heat treatment at 44 degrees C. Ku70/Ku80, but not Ku70 alone, could restore heat-inactivated DNA-PK.