Povirk Lawrence F, Zhou Rui-Zhe, Ramsden Dale A, Lees-Miller Susan P, Valerie Kristoffer
Department of Pharmacology and Toxicology, Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA.
Nucleic Acids Res. 2007;35(12):3869-78. doi: 10.1093/nar/gkm339. Epub 2007 May 25.
Previous work suggested that phosphorylation of DNA-PKcs at several serine/threonine (S/T) residues at positions 2609-2647 promotes DNA-PK-dependent end joining. In an attempt to clarify the role of such phosphorylation, end joining was examined in extracts of DNA-PKcs-deficient M059J cells. Joining of ends requiring gap filling prior to ligation was completely dependent on complementation of these extracts with exogenous DNA-PKcs. DNA-PKcs with either S/T --> A or S/T --> D substitutions at all six sites in the 2609-2647 cluster also supported end joining, but with markedly lower efficiency than wild-type protein. The residual end joining was greater with the S/T --> D-substituted than with the S/T --> A-substituted protein. A specific inhibitor of the kinase activity of DNA-PK, KU57788, completely blocked end joining promoted by wild type as well as both mutant forms of DNA-PK, while inhibition of ATM kinase did not. The fidelity of end joining was not affected by the mutant DNA-PKcs alleles or the inhibitors. Overall, the results support a role for autophosphorylation of the 2609-2647 cluster in promoting end joining and controlling the accessibility of DNA ends, but suggest that DNA-PK-mediated phosphorylation at other sites, on either DNA-PKcs or other proteins, is at least as important as the 2609-2647 cluster in regulating end joining.
先前的研究表明，DNA依赖蛋白激酶催化亚基（DNA-PKcs）在2609-2647位的几个丝氨酸/苏氨酸（S/T）残基发生磷酸化可促进DNA-PK依赖的末端连接。为了阐明这种磷酸化的作用，我们在DNA-PKcs缺陷的M059J细胞提取物中检测了末端连接情况。连接前需要填补缺口的末端连接完全依赖于用外源性DNA-PKcs对这些提取物进行互补。在2609-2647簇的所有六个位点具有S/T→A或S/T→D替换的DNA-PKcs也支持末端连接，但效率明显低于野生型蛋白。S/T→D替换的蛋白比S/T→A替换的蛋白残余末端连接效率更高。DNA-PK激酶活性的特异性抑制剂KU57788完全阻断了野生型以及两种突变形式的DNA-PK促进的末端连接，而抑制ATM激酶则没有这种作用。末端连接的保真度不受突变的DNA-PKcs等位基因或抑制剂的影响。总体而言，结果支持2609-2647簇的自磷酸化在促进末端连接和控制DNA末端可及性方面发挥作用，但表明DNA-PK介导的DNA-PKcs或其他蛋白质上其他位点的磷酸化在调节末端连接方面至少与2609-2647簇一样重要。
