Della Marina, Palmbos Phillip L, Tseng Hui-Min, Tonkin Louise M, Daley James M, Topper Leana M, Pitcher Robert S, Tomkinson Alan E, Wilson Thomas E, Doherty Aidan J
Cambridge Institute for Medical Research, University of Cambridge, Department of Haematology, Hills Road, Cambridge CB2 2XY, UK.
Science. 2004 Oct 22;306(5696):683-5. doi: 10.1126/science.1099824.
In mammalian cells, repair of DNA double-strand breaks (DSBs) by nonhomologous end-joining (NHEJ) is critical for genome stability. Although the end-bridging and ligation steps of NHEJ have been reconstituted in vitro, little is known about the end-processing reactions that occur before ligation. Recently, functionally homologous end-bridging and ligation activities have been identified in prokarya. Consistent with its homology to polymerases and nucleases, we demonstrate that DNA ligase D from Mycobacterium tuberculosis (Mt-Lig) possesses a unique variety of nucleotidyl transferase activities, including gap-filling polymerase, terminal transferase, and primase, and is also a 3' to 5' exonuclease. These enzyme activities allow the Mt-Ku and Mt-Lig proteins to join incompatible DSB ends in vitro, as well as to reconstitute NHEJ in vivo in yeast. These results demonstrate that prokaryotic Ku and ligase form a bona fide NHEJ system that encodes all the recognition, processing, and ligation activities required for DSB repair.
在哺乳动物细胞中，通过非同源末端连接（NHEJ）修复DNA双链断裂（DSB）对于基因组稳定性至关重要。尽管NHEJ的末端桥接和连接步骤已在体外重建，但对于连接前发生的末端加工反应却知之甚少。最近，在原核生物中发现了功能同源的末端桥接和连接活性。与其与聚合酶和核酸酶的同源性一致，我们证明来自结核分枝杆菌的DNA连接酶D（Mt-Lig）具有多种独特的核苷酸转移酶活性，包括缺口填充聚合酶、末端转移酶和引发酶，并且还是一种3'至5'核酸外切酶。这些酶活性使Mt-Ku和Mt-Lig蛋白能够在体外连接不相容的DSB末端，并在酵母体内重建NHEJ。这些结果表明，原核生物的Ku和连接酶形成了一个真正的NHEJ系统，该系统编码DSB修复所需的所有识别、加工和连接活性。
