Yumoto Y, Shirakawa H, Yoshida M, Suwa A, Watanabe F, Teraoka H
Department of Pathological Biochemistry, Medical Research Institute, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo 101-0062, Japan.
J Biochem. 1998 Sep;124(3):519-27. doi: 10.1093/oxfordjournals.jbchem.a022143.
The DNA-dependent protein kinase (DNA-PK) holoenzyme consists of a 470-kDa catalytic subunit (DNA-PKcs), a DNA-binding regulatory component known as Ku protein, and double-stranded DNA (dsDNA) with ends. We previously reported that the activity of DNA-PK in vitro is stimulated by non-histone chromosomal high mobility group proteins (HMG) 1 and 2 comprising two similar repeats, termed domains A and B, and an acidic C-terminal. Here we demonstrate that in vitro HMG1 and 2 can completely replace Ku protein as the DNA-binding regulatory component of DNA-PK. DNA-PKcs and Ku protein were separately purified from Raji nuclear extracts, and reconstituted into the DNA-PK holoenzyme in the presence of dsDNA. DNA-PKcs alone catalyzed DNA-dependent phosphorylation at a very low but significant level, and HMG1 and 2 markedly stimulated the phosphorylation of alpha-casein and a specific peptide substrate in a DNA-dependent manner. The HMG2-domains (A+B) polypeptide devoid of the C-terminal acidic region was more effective for DNA-PKcs stimulation than the full-length HMG2, and HMG2-domain A and -domain B polypeptides. Anti(Ku protein) antibodies inhibited the DNA-dependent phosphorylation activity of the DNA-PKcs:Ku protein complex, but not that of DNA-PKcs alone or when it was complexed with HMG1 or 2. These results demonstrate that HMG1 and 2 can function as the DNA-binding regulatory component for DNA-PKcs in vitro, and imply that a conformational change of dsDNA, which is elicited by regulatory components, is important for the stimulation of DNA-PK activity of DNA-PKcs.
DNA依赖性蛋白激酶（DNA-PK）全酶由一个470 kDa的催化亚基（DNA-PKcs）、一种称为Ku蛋白的DNA结合调节成分和带末端的双链DNA（dsDNA）组成。我们先前报道，体外DNA-PK的活性受到非组蛋白染色体高迁移率族蛋白（HMG）1和2的刺激，HMG 1和2包含两个相似的重复序列，称为结构域A和B以及一个酸性C末端。在此我们证明，体外HMG1和2可以完全替代Ku蛋白作为DNA-PK的DNA结合调节成分。DNA-PKcs和Ku蛋白分别从Raji细胞核提取物中纯化，并在dsDNA存在的情况下重构为DNA-PK全酶。单独的DNA-PKcs以非常低但显著的水平催化DNA依赖性磷酸化，而HMG1和2以DNA依赖性方式显著刺激α-酪蛋白和一种特异性肽底物的磷酸化。不含C末端酸性区域的HMG2-结构域（A+B）多肽比全长HMG2以及HMG2-结构域A和-结构域B多肽对DNA-PKcs的刺激更有效。抗（Ku蛋白）抗体抑制DNA-PKcs:Ku蛋白复合物的DNA依赖性磷酸化活性，但不抑制单独的DNA-PKcs或其与HMG1或2复合时的活性。这些结果表明，HMG1和2在体外可作为DNA-PKcs的DNA结合调节成分，并暗示由调节成分引发的dsDNA构象变化对刺激DNA-PKcs的DNA-PK活性很重要。
