Bhattarai Hitesh, Gupta Richa, Glickman Michael S
Weill Cornell Graduate School of Biomedical Sciences, New York, New York, USA.
Immunology Program, Sloan Kettering Institute, New York, New York, USA.
J Bacteriol. 2014 Oct;196(19):3366-76. doi: 10.1128/JB.01832-14. Epub 2014 Jun 23.
Nonhomologous end joining (NHEJ) is a recently described bacterial DNA double-strand break (DSB) repair pathway that has been best characterized for mycobacteria. NHEJ can religate transformed linear plasmids, repair ionizing radiation (IR)-induced DSBs in nonreplicating cells, and seal I-SceI-induced chromosomal DSBs. The core components of the mycobacterial NHEJ machinery are the DNA end binding protein Ku and the polyfunctional DNA ligase LigD. LigD has three autonomous enzymatic modules: ATP-dependent DNA ligase (LIG), DNA/RNA polymerase (POL), and 3' phosphoesterase (PE). Although genetic ablation of ku or ligD abolishes NHEJ and sensitizes nonreplicating cells to ionizing radiation, selective ablation of the ligase activity of LigD in vivo only mildly impairs NHEJ of linearized plasmids, indicating that an additional DNA ligase can support NHEJ. Additionally, the in vivo role of the POL and PE domains in NHEJ is unclear. Here we define a LigD ligase-independent NHEJ pathway in Mycobacterium smegmatis that requires the ATP-dependent DNA ligase LigC1 and the POL domain of LigD. Mycobacterium tuberculosis LigC can also support this backup NHEJ pathway. We also demonstrate that, although dispensable for efficient plasmid NHEJ, the activities of the POL and PE domains are required for repair of IR-induced DSBs in nonreplicating cells. These findings define the genetic requirements for a LigD-independent NHEJ pathway in mycobacteria and demonstrate that all enzymatic functions of the LigD protein participate in NHEJ in vivo.
非同源末端连接（NHEJ）是一种最近被描述的细菌DNA双链断裂（DSB）修复途径，在分枝杆菌中研究得最为深入。NHEJ能够重新连接转化的线性质粒，修复非复制细胞中电离辐射（IR）诱导的DSB，并封闭I-SceI诱导的染色体DSB。分枝杆菌NHEJ机制的核心成分是DNA末端结合蛋白Ku和多功能DNA连接酶LigD。LigD有三个自主的酶模块：ATP依赖性DNA连接酶（LIG）、DNA/RNA聚合酶（POL）和3'磷酸酯酶（PE）。虽然ku或ligD的基因敲除会消除NHEJ并使非复制细胞对电离辐射敏感，但在体内选择性敲除LigD的连接酶活性只会轻微损害线性化质粒的NHEJ，这表明另一种DNA连接酶可以支持NHEJ。此外，POL和PE结构域在NHEJ中的体内作用尚不清楚。在这里，我们在耻垢分枝杆菌中定义了一条不依赖LigD连接酶的NHEJ途径，该途径需要ATP依赖性DNA连接酶LigC1和LigD的POL结构域。结核分枝杆菌LigC也可以支持这种备用的NHEJ途径。我们还证明，虽然对于高效的质粒NHEJ来说是可有可无的，但POL和PE结构域的活性是修复非复制细胞中IR诱导的DSB所必需的。这些发现确定了分枝杆菌中不依赖LigD的NHEJ途径的遗传要求，并证明LigD蛋白的所有酶功能都参与了体内的NHEJ。
