Ma Yunmei, Schwarz Klaus, Lieber Michael R
Department of Pathology, Norris Comprehensive Cancer Center, Rm. 5428, University of Southern California Keck School of Medicine, Los Angeles, CA 90089-9176, USA.
DNA Repair (Amst). 2005 Jul 12;4(7):845-51. doi: 10.1016/j.dnarep.2005.04.013.
In eukaryotic cells, nonhomologous DNA end joining (NHEJ) is a major pathway for repair of double-strand DNA breaks (DSBs). Artemis and the 469kDa DNA-dependent protein kinase (DNA-PKcs) together form a key nuclease for NHEJ in vertebrate organisms. The structure-specific endonucleolytic activity of Artemis is activated by binding to and phosphorylation by DNA-PKcs. We tested various DNA structures in order to understand the range of structural features that are recognized by the Artemis:DNA-PKcs complex. We find that all tested substrates that contain single-to-double-strand transitions can be cleaved by the Artemis:DNA-PKcs complex near the transition region. The cleaved substrates include heterologous loops, stem-loops, flaps, and gapped substrates. Such versatile activity on single-/double-strand transition regions is important in understanding how reconstituted NHEJ systems that lack DNA polymerases can join incompatible DNA ends and yet preserve 3' overhangs. Additionally, the flexibility of the Artemis:DNA-PKcs nuclease may be important in removing secondary structures that hinder processing of DNA ends during NHEJ.
在真核细胞中，非同源DNA末端连接（NHEJ）是双链DNA断裂（DSB）修复的主要途径。Artemis与469kDa的DNA依赖性蛋白激酶（DNA-PKcs）共同构成脊椎动物中NHEJ的关键核酸酶。Artemis的结构特异性核酸内切酶活性通过与DNA-PKcs结合并被其磷酸化而激活。为了了解Artemis:DNA-PKcs复合物识别的结构特征范围，我们测试了各种DNA结构。我们发现，所有测试的包含单链到双链转变的底物都可以被Artemis:DNA-PKcs复合物在转变区域附近切割。切割的底物包括异源环、茎环、翼片和有缺口的底物。这种在单/双链转变区域的多功能活性对于理解缺乏DNA聚合酶的重组NHEJ系统如何连接不相容的DNA末端并保留3'突出端非常重要。此外，Artemis:DNA-PKcs核酸酶的灵活性对于去除在NHEJ过程中阻碍DNA末端加工的二级结构可能很重要。
