Loss of the catalytic subunit of the DNA-dependent protein kinase in DNA double-strand-break-repair mutant mammalian cells.

The DNA-dependent protein kinase (DNA-PK) consists of three polypeptide components: Ku-70, Ku-80, and an approximately 350-kDa catalytic subunit (p350). The gene encoding the Ku-80 subunit is identical to the x-ray-sensitive group 5 complementing gene XRCC5. Expression of the Ku-80 cDNA rescues both DNA double-strand break (DSB) repair and V(D)J recombination in group 5 mutant cells. The involvement of Ku-80 in these processes suggests that the underlying defect in these mutant cells may be disruption of the DNA-PK holoenzyme. In this report we show that the p350 kinase subunit is deleted in cells derived from the severe combined immunodeficiency mouse and in the Chinese hamster ovary cell line V-3, both of which are defective in DSB repair and V(D)J recombination. A centromeric fragment of human chromosome 8 that complements the scid defect also restores p350 protein expression and rescues in vitro DNA-PK activity. These data suggest the scid gene may encode the p350 protein or regulate its expression and are consistent with a model whereby DNA-PK is a critical component of the DSB-repair pathway. IMAGES:

依赖DNA的蛋白激酶 (DNA-dependent protein kinase, DNA-PK)由三种多肽组分构成：Ku-70、Ku-80以及分子量约350 kDa的催化亚基 (p350)。编码Ku-80亚基的基因与X射线敏感型5组互补基因XRCC5完全一致。Ku-80的互补DNA (complementary DNA, cDNA)的表达，可恢复5组突变细胞的DNA双链断裂 (double-strand break, DSB)修复能力与V(D)J重组功能。Ku-80在上述过程中的参与性表明，此类突变细胞的潜在缺陷可能为DNA-PK全酶的功能紊乱。本研究显示，源自重症联合免疫缺陷 (severe combined immunodeficiency, SCID)小鼠的细胞，以及中国仓鼠卵巢细胞系V-3，均存在p350激酶亚基的缺失；而这两类细胞均存在DSB修复与V(D)J重组的功能缺陷。可互补SCID缺陷的人类8号染色体着丝粒片段，同样能够恢复p350蛋白的表达，并修复体外实验中的DNA-PK活性。上述数据表明，SCID基因可能编码p350蛋白，或对其表达进行调控；这一结果与“DNA-PK是DSB修复通路关键组分”的模型相一致。图片：