Woods Derek S, Sears Catherine R, Turchi John J
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.
Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.
PLoS One. 2015 May 15;10(5):e0127321. doi: 10.1371/journal.pone.0127321. eCollection 2015.
DNA double strand breaks (DSBs) can be generated by endogenous cellular processes or exogenous agents in mammalian cells. These breaks are highly variable with respect to DNA sequence and structure and all are recognized in some context by the DNA-dependent protein kinase (DNA-PK). DNA-PK is a critical component necessary for the recognition and repair of DSBs via non-homologous end joining (NHEJ). Previously studies have shown that DNA-PK responds differentially to variations in DSB structure, but how DNA-PK senses differences in DNA substrate sequence and structure is unknown. Here we explore the enzymatic mechanisms by which DNA-PK is activated by various DNA substrates and provide evidence that the DNA-PK is differentially activated by DNA structural variations as a function of the C-terminal region of Ku80. Discrimination based on terminal DNA sequence variations, on the other hand, is independent of the Ku80 C-terminal interactions and likely results exclusively from DNA-dependent protein kinase catalytic subunit interactions with the DNA. We also show that sequence differences in DNA termini can drastically influence DNA repair through altered DNA-PK activation. These results indicate that even subtle differences in DNA substrates influence DNA-PK activation and ultimately the efficiency of DSB repair.
在哺乳动物细胞中，DNA双链断裂（DSB）可由内源性细胞过程或外源性因素产生。这些断裂在DNA序列和结构方面具有高度变异性，并且在某些情况下都会被DNA依赖性蛋白激酶（DNA-PK）识别。DNA-PK是通过非同源末端连接（NHEJ）识别和修复DSB所必需的关键组分。先前的研究表明，DNA-PK对DSB结构的变化有不同反应，但DNA-PK如何感知DNA底物序列和结构的差异尚不清楚。在这里，我们探讨了DNA-PK被各种DNA底物激活的酶促机制，并提供证据表明DNA-PK作为Ku80 C末端区域的功能被DNA结构变异差异激活。另一方面，基于末端DNA序列变异的识别独立于Ku80 C末端相互作用，并且可能完全由DNA依赖性蛋白激酶催化亚基与DNA的相互作用导致。我们还表明，DNA末端的序列差异可通过改变DNA-PK激活来极大地影响DNA修复。这些结果表明，即使DNA底物中的细微差异也会影响DNA-PK激活，并最终影响DSB修复的效率。
