Abstract
The Ku protein, a heterodimer composed of Ku70 and Ku 86, was first found while studying radiation-sensitive ( IRs ) cell lines. Scientists discovered that rodent cells became sensitive to radiation when they are deficient in Ku70 or Ku86. It seems that Ku protein is involved in repair of DNA double-strand breaks. In addition, Ku protein takes part in composition of DNA-dependent protein kinase (DNA-PK) with DNA-PKcs (catalytic subunit). The kinase has been shown to phosphorylate proteins, which are involved in repair, replication and transcription in vitro. Ku also plays an important role in repair, replication, transcription and V (D) J recombination. Therefore, it is important to have a better understanding of the characteristics and functions of Ku.The most important function of Ku is the DNA-end binding (DEB) activity. Based on this reason, our laboratory focuses on studying this special activity of KuFirst, by using three different cell lines, we found two DEB proteins present in human diploid fibroblasts (HDF), but only one in RKO and HeLa cells. The extra DEB protein in HF cells migrated faster than Ku protein in gels for DEB assays. When cells became old after continuous subculturing, the whole cell extract (WCE) only showed the faster-migrating DNA-protein complex. In Western blotting, a 86 and 69 kDa protein were detected in WCE of young HFs, but only the 69 kDa protein was revealed in WCE of old HFs. At the same time, Ku70 subunit was found to have the same level in both extracts. After series of mixing experiments, the results suggest that a protease capable of cleaving Ku is present in higher amount in old HFs than in young HFs, and is absent in HeLa cells. We have tried to test several protease inhibitors and found that leupeptin can decrease the cleavage of Ku protein effectively.Although this protease from HF cells has been proved to be able to cleavage Ku protein and it seems to be activated more strongly in older cells, it can not effect Ku protein in vivo. Therefore, how and why this protease works in cells needs to be further studied.Second, we used radiation inactivation to measure the functional size of Ku protein responsible for DEB activity. The result showed that the size of Ku binding to DNA ends was 300 kDa, whether the length of the probes is 222 bp or 25 bp. We suggest that two Ku proteins bind to one DNA end, so that functional size is about twice of that of Ku (86kDa +70kDa). If this proves to be true, it will give Ku a whole new definition.