The pathways and outcomes of mycobacterial NHEJ depend on the structure of the broken DNA ends
The pathways and outcomes of mycobacterial NHEJ depend on the structure of the broken DNA ends
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DOI：
10.1101/gad.1631908
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发表时间：
2008-02-15
影响因子：
10.5
通讯作者：
Shuman, Stewart
中科院分区：
文献类型：
--
作者：
Aniukwu, Jideofor;Glickman, Michael S.;Shuman, Stewart
分枝杆菌可以通过非同源末端连接（NHEJ）系统修复DNA双链断裂（DSB），该系统包括专用DNA连接酶（LigD）和DNA末端结合蛋白Ku。在这里，我们利用一种改进的基于质粒的NHEJ测定和一组在Ku或DNA连接酶中具有缺失或突变的耻垢分枝杆菌菌株来询问LigD的三种催化活性（聚合酶、连接酶和3'磷酸酯酶）和结构域（POL、LIG和PE）对体内NHEJ的效率和分子结果的贡献。通过平行分析平端、5'突出端和3'突出端DSB的修复，我们发现了一种新的末端连接途径，该途径特异于具有3'突出端的断裂，其是Ku和LigD独立的并且完全忠实。这种3'突出端NHEJ途径不依赖于连接酶B和C;我们推测它依赖于NAD（+）依赖性LigA，即必需的复制连接酶。我们发现平端和5'突出端DSB的有效修复严格依赖于Ku和LigD POL结构域，但不依赖于LigD聚合酶活性，其主要用于促进NHEJ不忠实。PE失活LigD突变对NHEJ结果的影响的缺乏，特别是在钝端或5'突出端断裂处的缺失和插入之间的平衡，反对LigD是缺失形成的催化剂。连接酶失活LigD突变（或LIG结构域的缺失）对钝端和5'突出端DSB修复的效率具有适度的影响，因为链密封活性可以由其他驻留的ATP依赖性连接酶之一（可能是LigC）反式提供。对备用连接酶的依赖伴随着在平端和5'突出端DSB修复期间保真度的急剧损失。我们得出结论，分枝杆菌NHEJ的机制是多种多样的，其结果取决于DSB的初始结构以及末端加工和末端密封组件的可用集合，而不仅仅限于Ku和LigD。
Mycobacteria can repair DNA double-strand breaks (DSBs) via a nonhomologous end-joining (NHEJ) system that includes a dedicated DNA ligase (LigD) and the DNA end-binding protein Ku. Here we exploit an improved plasmid-based NHEJ assay and a collection of Mycobacterium smegmatis strains bearing deletions or mutations in Ku or the DNA ligases to interrogate the contributions of LigD's three catalytic activities (polymerase, ligase, and 3' phosphoesterase) and structural domains (POL, LIG, and PE) to the efficiency and molecular outcomes of NHEJ in vivo. By analyzing in parallel the repair of blunt, 5' overhang, and 3' overhang DSBs, we discovered a novel end-joining pathway specific to breaks with 3' overhangs that is Ku- and LigD-independent and perfectly faithful. This 3' overhang NHEJ pathway is independent of ligases B and C; we surmise that it relies on NAD(+)-dependent LigA, the essential replicative ligase. We find that efficient repair of blunt and 5' overhang DSBs depends stringently on Ku and the LigD POL domain, but not on the LigD polymerase activity, which mainly serves to promote NHEJ infidelity. The lack of an effect of PE-inactivating LigD mutations on NHEJ outcomes, especially the balance between deletions and insertions at blunt or 5' overhang breaks, argues against LigD being the catalyst of deletion formation. Ligase-inactivating LigD mutations (or deletion of the LIG domain) have a modest impact on the efficiency of blunt and 5' overhang DSB repair, because the strand sealing activity can be provided in trans by one of the other resident ATP-dependent ligases (likely LigC). Reliance on the backup ligase is accompanied by a drastic loss of fidelity during blunt end and 5' overhang DSB repair. We conclude that the mechanisms of mycobacterial NHEJ are many and the outcomes depend on the initial structures of the DSBs and the available ensemble of end-processing and end-sealing components, which are not limited to Ku and LigD.