Abstract
DNA double strand break (DSB) is the major lesion leading to cell lethality by ionizing radiation (IR) and its repair is important in maintaining the genetic integrity of the genome. Accumulated evidence in recent years indicates that Ku is involved in DNA DSB rejoining and V(D)J recombination. Ku is an abundant nuclear DNA end-binding (DEB) protein composed of 70 and 86 kDa subunits, and is the regulatory component of the DNA-dependent protein kinase (DNA- PK) . The 350 kDa (p350) catalytic subunit of DNA-PK phosphorylates several transcription factors in vitro. The precise cellular role of Ku is unknown, but it has been implicated in both DNA replication and repair and in transcriptional control. We have been interested in the detail of the DEB activity ofKu in order to understand its biological roles. In this study, we attempt to find the possible regulatory mechanism of DEB activity of Ku. We found the DEB activity could be regulated by serum in CHO Kl cells, because the activity of the S and M phase was about six-fold lower than the G1 and control population when serum starvation was used to synchronize cells. In comparison, centrifugal elutriation experiments indicated that DEB activity was the lowest in M phase and the highest in S phase in K1, HeLa, and RKO cell lines. Mixing experiments showed the changes of DEB activity in the different stages of the cell cycle was not due to a trans-acting stimulator or inhibitor. Unexpectedly, after exposure to X-irradiation, the activity in extracts of RKO cells did not change significantly within 1 h, however, the activity in serum-starved S phase K1 cells was elevated by IR. The stimulation by IR was specific since other DNA damage agents (UV, EMS) did not regulate DEB activity. RKO cells were utilized to study the mechanism of decreased DEB activity in M phase cells. By Western analysis, the DEB activity in G1 and S phase of the elutriated RKO cells correlated well with the amount of Ku protein. However, the level of Ku protein in M phase cells was not obviously less than the G1 and S phase cells although the DEB activity was decreased significantly. Several inhibitors of protein kinases and phosphatase were used in vivo to show that DEB activity in M phase cells was regulated by phosphorylation/dephosphorylation. Interestingly, immunoprecipitation of the 32P-orthophosphate l led proteins in RKO cells failed to show Ku70 or Ku80 was phosphorylated. Several hypothesis were proposed to explain the unexpected discrepancy.