Gu X Y, Weinfeld M A, Povirk L F
Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298, USA.
Biochemistry. 1998 Jul 7;37(27):9827-35. doi: 10.1021/bi980198o.
Previous work with Xenopus egg extracts suggested that a wortmannin-sensitive protein phosphorylation event precedes both the removal of modified termini from DNA double-strand break ends and the joining of unmodified ends. To assess the possible role of DNA-dependent protein kinase in effecting this phosphorylation, both DNA end-joining and DNA-stimulated phosphorylation were examined in the presence of various inhibitors. Linear but not supercoiled DNA stimulated the phosphorylation of several endogenous proteins in the extracts, including species of approximately 48, 87, and 96 kDa. This phosphorylation was selectively suppressed by the kinase inhibitors wortmannin, dimethylaminopurine, and LY294002, with a dose response that in each case paralleled the inhibition of DNA end-joining. If wortmannin was added while the end-joining reaction was in progress, end-joining of DNA already present in the reaction continued for some time, but newly added DNA was not joined or processed at all. Ends with 3'-hydroxyl termini were joined much faster than those with 3'-phosphoglycolate termini, although both were equally effective in stimulating protein phosphorylation. The results support a role for DNA-dependent protein kinase in regulating end-joining in vitro, and suggest that at least one of the necessary phosphorylations involves a protein bound at or near the DNA end to be joined. In contrast, nuclear extracts from human cells joined double-strand breaks with normal but not modified termini, and the joining was unaffected by kinase inhibitors, suggesting that the dominant mechanism of end-joining in these extracts did not involve DNA-PK.
之前对非洲爪蟾卵提取物的研究表明，渥曼青霉素敏感的蛋白磷酸化事件先于从DNA双链断裂末端去除修饰末端以及未修饰末端的连接。为了评估DNA依赖性蛋白激酶在实现这种磷酸化过程中可能发挥的作用，在存在各种抑制剂的情况下检测了DNA末端连接和DNA刺激的磷酸化。线性而非超螺旋DNA刺激了提取物中几种内源性蛋白的磷酸化，包括分子量约为48、87和96 kDa的蛋白。这种磷酸化被激酶抑制剂渥曼青霉素、二甲基氨基嘌呤和LY294002选择性抑制，每种抑制剂的剂量反应都与DNA末端连接的抑制情况平行。如果在末端连接反应进行时加入渥曼青霉素，反应中已存在的DNA的末端连接会持续一段时间，但新加入的DNA根本不会被连接或加工。具有3'-羟基末端的末端比具有3'-磷酸乙醇酸末端的末端连接速度快得多，尽管两者在刺激蛋白磷酸化方面同样有效。这些结果支持了DNA依赖性蛋白激酶在体外调节末端连接中的作用，并表明至少一种必要的磷酸化涉及与待连接的DNA末端或其附近结合的蛋白。相比之下，人细胞核提取物能连接具有正常而非修饰末端的双链断裂，且连接不受激酶抑制剂的影响，这表明这些提取物中末端连接的主要机制不涉及DNA-PK。
