Departments of Pathology, Biochemistry, Molecular Microbiology & Immunology, and Section of Molecular & Computational Biology (Department of Biological Sciences), University of Southern California, Los Angeles, CA, 90089-9176, USA.
Departments of Pathology, Biochemistry, Molecular Microbiology & Immunology, and Section of Molecular & Computational Biology (Department of Biological Sciences), University of Southern California, Los Angeles, CA, 90089-9176, USA.
Prog Biophys Mol Biol. 2023 Jul-Aug;180-181:105-119. doi: 10.1016/j.pbiomolbio.2023.05.001. Epub 2023 May 5.
Cellular and biochemical studies of nonhomologous DNA end joining (NHEJ) have long established that nuclease and polymerase action are necessary for the repair of a very large fraction of naturally-arising double-strand breaks (DSBs). This conclusion is derived from NHEJ studies ranging from yeast to humans and all genetically-tractable model organisms. Biochemical models derived from recent real-time and structural studies have yet to incorporate physical space or timing for DNA end processing. In real-time single molecule FRET (smFRET) studies, we analyzed NHEJ synapsis of DNA ends in a defined biochemical system. We described a Flexible Synapsis (FS) state in which the DNA ends were in proximity via only Ku and XRCC4:DNA ligase 4 (X4L4), and in an orientation that would not yet permit ligation until base pairing between one or more nucleotides of microhomology (MH) occurred, thereby allowing an in-line Close Synapsis (CS) state. If no MH was achievable, then XLF was critical for ligation. Neither FS or CS required DNA-PKcs, unless Artemis activation was necessary to permit local resection and subsequent base pairing between the two DNA ends being joined. Here we conjecture on possible 3D configurations for this FS state, which would spatially accommodate the nuclease and polymerase processing steps in an iterative manner. The FS model permits repeated attempts at ligation of at least one strand at the DSB after each round of nuclease or polymerase action. In addition to activation of Artemis, other possible roles for DNA-PKcs are discussed.
非同源 DNA 末端连接 (NHEJ) 的细胞和生化研究长期以来一直表明，核酸酶和聚合酶的作用对于修复大量自然产生的双链断裂 (DSB) 是必要的。这一结论来自于从酵母到人以及所有遗传上可操作的模式生物的 NHEJ 研究。最近的实时和结构研究得出的生化模型尚未纳入 DNA 末端处理的物理空间或时间。在实时单分子荧光共振能量转移 (smFRET) 研究中，我们在一个定义明确的生化系统中分析了 DNA 末端的 NHEJ 连接。我们描述了一种灵活连接 (FS) 状态，其中 DNA 末端仅通过 Ku 和 XRCC4:DNA 连接酶 4 (X4L4) 接近，并且处于一种取向，直到一个或多个微同源性 (MH) 的核苷酸之间发生碱基配对，才能允许进行连接，从而允许进行在线紧密连接 (CS) 状态。如果无法实现 MH，则 XLF 对于连接至关重要。FS 或 CS 都不需要 DNA-PKcs，除非 Artemis 激活对于允许两个连接的 DNA 末端之间的局部切除和随后的碱基配对是必要的。在这里，我们推测这种 FS 状态的可能 3D 构型，它将以迭代方式在空间上容纳核酸酶和聚合酶的处理步骤。FS 模型允许在每个核酸酶或聚合酶作用循环之后，至少在 DSB 的一条链上重复尝试连接。除了 Artemis 的激活外，还讨论了 DNA-PKcs 的其他可能作用。
