Ku86 Defines the Genetic Defect and Restores X-Ray Resistance and V(D)J Recombination to Complementation Group 5 Hamster Cell Mutants

Abstract

X-ray-sensitive hamster cells in complementation groups 4, 5, 6, and 7 are impaired for both double-strand break repair and V(D)J recombination. Here we show that in two mutant cell lines (XR-V15B and XR-V9B) from group 5, the genetic defects are in the gene encoding the 86-kDa subunit of the Ku autoantigen, a nuclear protein that binds to double-stranded DNA ends. These mutants express Ku86 mRNA containing deletions of 138 and 252 bp, respectively, and the encoded proteins contain internal, in-frame deletions of 46 and 84 amino acids. Two X-ray-resistant revertants of XR-V15B expressed two Ku86 transcripts, one with and one without the deletion, suggesting that reversion occurred by activation of a silent wild-type allele. Transfection of fulllength cDNA encoding hamster Ku86 into XR-V15B cells resulted in a complete rescue of DNA-end-binding (DEB) activity and Ku70 levels, suggesting that Ku86 stabilizes the Ku70 polypeptide. In addition, cells expressing wild-type levels of DEB activity were fully rescued for X-ray resistance and V(D)J recombination, whereas cells expressing lower levels of DEB activity were only partially rescued. Thus, Ku is an essential component of the pathway(s) utilized for the resolution of DNA double-strand breaks induced by either X rays or V(D)J recombination, and mutations in the Ku86 gene are responsible for the phenotype of group 5 cells.All cells possess a mechanism for repairing DNA doublestrand breaks (DSBs) produced by ionizing radiation. Cells of the immune system must also resolve DNA DSBs produced by V(D)J recombination of the immunoglobulin and T-cell receptor genes during development of B and T cells (reviewed in reference 28). In fact, the biochemical pathways for DSB repair and V(D)J recombination have a number of common factors. Evidence of this was first described for the severe combined immune deficient (scid) mouse, which is hypersensitive to ionizing radiation because of defective DSB repair (1, 12, 18) and immune deficient because of defective V(D)J recombination (29). Subsequently, other X-ray-sensitive rodent cell lines defective in DSB repair were also found to have impaired V(D)J recombination (17,27,34,45,47). These cell lines fall into four complementation groups, 4, 5, 6, and 7, with group 7 corresponding to the scid defect (43, 52). The human genes capable of rescuing these mutants are designated XRCC4, XRCC5, XRCC6, and XRCC7, respectively (XRCC denotes X ray cross-complementing) (for a review, see reference 51).Recently, it has been shown that the Ku protein is involved in both DSB repair and V(D)J recombination. Ku is an abundant nuclear protein identified originally as an autoantigen recognized by sera from patients with autoimmune diseases, including scleroderma-polymyositis overlap syndrome and systemic lupus erythematosus (31

Ku80 can translocate to the nucleus independent of the translocation of Ku70 using its own nuclear localization signal

“…This agreed well with the results obtained by immunocytochemical analysis (Reeves, 1985;Yaneva et al, 1985;Mimori et al, 1986;Koike et al, 1998). Since both Ku70 and Ku80 monomers are highly unstable compared to their heterodimers (Errami et al, 1996;Gu et al, 1997;Singleton et al, 1997), we were interested in studying the nuclear translocation of Ku80 and its heterodimer partner Ku70. Osipovich et al (1997) reported that mutations in Ku80 corresponding to amino acids 453 and 454 abolished its ability to interact with Ku70, using an in vitro binding assay and the yeast two-hybrid analysis.…”

“…Both Ku70 and Ku80 are highly unstable when expressed individually (Satoh et al, 1995). Loss of one of the Ku subunits results in a signi®cant decrease in the steady-state level of the other (Errami et al, 1996;Gu et al, 1997;Singleton et al, 1997). These ®ndings strongly suggest that Ku70 and Ku80 function as heterodimers.…”

Transfer of Ku86 RNA antisense decreases the radioresistance of human fibroblasts

“…31,32 In rodents, Ku DNA binding represents the predominant, if only, mechanism for DNA-PK activation, because the Ku-defective mutants have undetectable DNA-PK activity. 33 Also in rodents, the DNA-PK complex has been shown to play an important role in the response to ionizing radiation, because mutations in one of the DNA-PK subunits lead to an extreme x-ray-sensitivity along with a deficiency in both DSB repair and V(D)J recombination, 34,35 suggesting that the integrity of this complex is necessary for a normal enzymatic activity. It was also possible to complement the deficient phenotype of DNA-PK mutant rodent cell lines by transferring the corresponding genes for Ku86 and DNA-PKcs.…”

“…It was also possible to complement the deficient phenotype of DNA-PK mutant rodent cell lines by transferring the corresponding genes for Ku86 and DNA-PKcs. [35][36][37] The role of DNA-PK complex in the radiosensitivity of human cells is still being investigated. Interestingly, DNA-PKcs presents a sequence homology in the carboxyl-terminal domain with the ataxia telangiectasia gene (ATM) corresponding to the phosphatidylinositol 3-kinase superfamily catalytic domain.…”

Activation of PARP-1 in response to bleomycin depends on the Ku antigen and protein phosphatase 5

“…To determine whether Ku might also be important for the activation of PARP-1 subsequent to the DSBs induced by bleomycin, we compared the activity of PARP-1 after treatment in extracts prepared from V79 cells, a hamster embryonic lung fibroblast cell line and V15B cells, a line clonally derived from V79 containing a mutation in the Ku80 gene that leads to the absence of Ku70 and Ku80 (Errami et al, 1996). Typical strong activation of PARP-1 activity was observed in extracts prepared from V79 cells and the response included a shifting and spreading of the PARP-1 band on immunoblots ( Figure 3a).…”