Mechanisms of DNA double strand break repair and chromosome aberration formation
Mechanisms of DNA double strand break repair and chromosome aberration formation
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DOI：
10.1159/000077461
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发表时间：
2004-01-01
影响因子：
1.7
通讯作者：
Pantelias, G
中科院分区：
文献类型：
--
作者：
Iliakis, G;Wang, H;Pantelias, G
DNA双链断裂（DSB）的未修复或错误修复是导致染色体畸变的重要原因。在高等真核生物的DNA中由内源性过程或外源性试剂诱导的DSB原则上可以通过非同源末端连接（NHEJ）或同源定向修复（HDR）来修复。选择DSB修复途径的基础仍然未知，但可能取决于诱导剂或过程。评估NHEJ和HDR对电离辐射（IR）诱导的DSB修复的相对贡献对于我们理解导致染色体畸变形成的机制非常重要。在这里，我们回顾了我们的实验室最近的工作，有助于这条调查线。使用脉冲场凝胶电泳分析辐照细胞中的DSB再连接，揭示了一个快速的组分，半衰期为10-30 min。在DNA-PKcs、Ku、DNA连接酶IV或XRCC 4突变的细胞中，以及在DNA-PK化学抑制后，DSB再连接的该组分严重受损，表明它反映了经典的NHEJ;我们将这种形式的DSB再连接D-NHEJ称为DSB再连接D-NHEJ，以表示其对DNA-PK的依赖性。尽管任何这些因素的化学抑制或突变都会延迟处理，但细胞最终会使用以较慢动力学（半衰期2-10 h）运行的替代途径去除大部分DSB。这种替代的，缓慢的DSB重新连接的途径在缺乏几个基因的RAD 52上位性组的突变体中不受影响，这表明它可能不反映HDR。我们提出，它反映了NHEJ的替代形式，作为DNA-PK依赖性（D-NHEJ）途径的备份（B-NHEJ）。生物化学研究证实了在没有DNA-PK的情况下，细胞提取物中存在DNA末端连接活性，并表明D-NHEJ在活性时起主导作用。总的来说，这些观察结果表明，NHEJ，通过两个互补的途径，B-NHEJ和D-NHEJ，是主要的机制，通过它IR诱导的DSB从高等真核生物的DNA中删除。HDR被认为要么作用于一小部分IR诱导的DSB，要么在初始端部连接后的步骤中参与修复过程。我们认为，高速D-NHEJ是高等真核生物中围绕新进化的DNA-PKcs和预先存在的因子精心安排的进化发展。它通过优化的突触机制在几分钟内恢复染色体的完整性，该机制通过染色质和核基质中的蛋白质-蛋白质相互作用序列进行操作。因此，D-NHEJ主要连接正确的DNA末端并抑制染色体畸变的形成，尽管不能确保断裂周围的DNA序列的恢复。B-NHEJ可能是一种进化上较老的途径，其突触机制较不优化，以数小时的动力学重新连接DNA末端。B-NHEJ的缓慢动力学和次优突触机制允许通过连接不正确的末端进行更多的时间交换，并导致野生型和D-NHEJ突变细胞中染色体畸变的形成。版权所有（C）2003 S. Karger AG，巴塞尔。
It is widely accepted that unrepaired or misrepaired DNA double strand breaks (DSBs) lead to the formation of chromosome aberrations. DSBs induced in the DNA of higher eukaryotes by endogenous processes or exogenous agents can in principle be repaired either by non-homologous endjoining (NHEJ), or homology directed repair (HDR). The basis on which the selection of the DSB repair pathway is made remains unknown but may depend on the inducing agent, or process. Evaluation of the relative contribution of NHEJ and HDR specifically to the repair of ionizing radiation (IR) induced DSBs is important for our understanding of the mechanisms leading to chromosome aberration formation. Here, we review recent work from our laboratories contributing to this line of inquiry. Analysis of DSB rejoining in irradiated cells using pulsed-field gel electrophoresis reveals a fast component operating with half times of 10-30 min. This component of DSB rejoining is severely compromised in cells with mutations in DNA-PKcs, Ku, DNA ligase IV, or XRCC4, as well as after chemical inhibition of DNA-PK, indicating that it reflects classical NHEJ; we termed this form of DSB rejoining D-NHEJ to signify its dependence on DNA-PK. Although chemical inhibition, or mutation, in any of these factors delays processing, cells ultimately remove the majority of DSBs using an alternative pathway operating with slower kinetics (half time 2-10 h). This alternative, slow pathway of DSB rejoining remains unaffected in mutants deficient in several genes of the RAD52 epistasis group, suggesting that it may not reflect HDR. We proposed that it reflects an alternative form of NHEJ that operates as a backup (B-NHEJ) to the DNA-PK-dependent (D-NHEJ) pathway. Biochemical studies confirm the presence in cell extracts of DNA end joining activities operating in the absence of DNA-PK and indicate the dominant role for D-NHEJ, when active. These observations in aggregate suggest that NHEJ, operating via two complementary pathways, B-NHEJ and D-NHEJ, is the main mechanism through which IR-induced DSBs are removed from the DNA of higher eukaryotes. HDR is considered to either act on a small fraction of IR induced DSBs, or to engage in the repair process at a step after the initial end joining. We propose that high speed D-NHEJ is an evolutionary development in higher eukaryotes orchestrated around the newly evolved DNA-PKcs and pre-existing factors. It achieves within a few minutes restoration of chromosome integrity through an optimized synapsis mechanism operating by a sequence of protein-protein interactions in the context of chromatin and the nuclear matrix. As a consequence D-NHEJ mostly joins the correct DNA ends and suppresses the formation of chromosome aberrations, albeit, without ensuring restoration of DNA sequence around the break. B-NHEJ is likely to be an evolutionarily older pathway with less optimized synapsis mechanisms that rejoins DNA ends with kinetics of several hours. The slow kinetics and suboptimal synapsis mechanisms of B-NHEJ allow more time for exchanges through the joining of incorrect ends and cause the formation of chromosome aberrations in wild type and D-NHEJ mutant cells. Copyright (C) 2003 S. Karger AG, Basel.