Mechanism of DNA interstrand crosslink repair in vivo

 描述（由申请人提供）：DNA链间交联是一种特别致命的DNA损伤形式，代表了对复制和转录的绝对阻断。形成交联的化学品在自然界中被发现时已被证明是高毒性的，在特定癌症中作为化疗剂是独特有效的，并且对涉及增生或增生性病症的一系列疾病状态是有效的治疗。 虽然已经分离出几个基因，当突变时，使细胞对交联高度敏感，但这些复杂病变如何在细胞中修复和处理的许多方面仍然未知。我们所知道的大部分关于链间交联修复来自真核生物提取物的研究，并表明复制依赖和复制无关的机制都存在。这两种机制都涉及多个修复途径，核苷酸切除修复与重组，translesion合成，以及其他替代核酸酶复合物的耦合组件。然而，在最初的切口事件之后，所有这些模型仍然是高度推测性的，并且受到重建这种多步骤、多途径修复过程的挑战以及哺乳动物细胞中可用的细胞测定的复杂性和缺乏的阻碍。 在这里，我们建议直接识别的细胞通路和结构中间体，在体内使用大肠杆菌的模式生物，其中复制和修复过程是高度保守的链间交联修复过程中出现的。在大肠大肠杆菌，我们已经建立了独特的细胞检测，以监测复制叉处理和全球修复这些病变。此外，在初步数据中，我们表明，我们已经确定了一种替代方案， 核酸内切酶，类似于哺乳动物细胞，其与核苷酸切除修复复合物偶联并且对于交联修复是重要的。这些试验将使我们能够直接和明确地确定在体内交联修复过程中出现的修复和进行性中间体。我们描述了将实现的三个目标。1)我们将鉴定出参与修复大肠杆菌DNA链间交联的基因。大肠杆菌中，并确定它们是否在体内复制依赖或复制独立（全局基因组）修复途径。2)我们将在体内鉴定与DNA交联的非复制依赖性修复相关的细胞中间产物和生化途径。3)我们将在体内鉴定与DNA交联的复制依赖性修复相关的细胞中间产物和生化途径。 这些研究的结果将确定在体内修复这种医学相关病变的途径，并可能提出新的治疗方法，利用这些病变治疗癌症和其他过度增殖性疾病。

 DESCRIPTION (provided by applicant): DNA interstrand crosslinks are a particularly lethal form of DNA damage that represent an absolute block to replication and transcription. Chemicals forming crosslinks have proven to be highly toxic when found in nature, uniquely potent as chemotherapeutics in specific cancers, and effective treatments for a range of diseases states involving hyperplastic or displastic conditions. Although several genes have been isolated that, when mutated, render cells hypersensitive to crosslinks, many aspects of how these complex lesions are repaired and processed in cells remain unknown. Much of what we know about interstrand crosslink repair has come from eukaryotic studies in extracts and suggest that both replication-dependent and replication-independent mechanisms exist. Both mechanisms are proposed to involve multiple repair pathways, coupling components of nucleotide excision repair with recombination, translesion synthesis, as well as other alternative nuclease complexes. However, after the initial incision event, all these models remain highly speculative, and are hampered by the challenges of reconstituting this multi-step, multi-pathway repair process as well as by the complexity and lack of cellular assays available in mammalian cells. Here, we propose to directly identify the cellular pathways and structural intermediates that arise during the repair of interstrand crosslinks in vivo using the model organism of E.coli, where the processes of replication and repair are highly conserved. In E. coli, we have established unique cellular assays to monitor the replication fork processing and global repair for these lesions. In addition, in preliminary data, we show that we have identified an alternative endonuclease, similar to mammalian cells, that couples with the nucleotide excision repair complex and is important for crosslink repair. These assays will allow us to directly and definitively identify the repair and progressive intermediates that arise during crosslink repair i vivo We describe three aims that will be accomplished. 1) We will identify the genes involved in repairing DNA interstrand crosslinks in E. coli and determine whether they operate in a replication-dependent or replication-independent (global genomic) repair pathway in vivo. 2) We will identify the cellular intermediates and biochemical pathway associated with the replication-independent repair of DNA crosslinks in vivo. 3) We will identify the cellular intermediates and biochemical pathway associated with the replication-dependent repair of DNA crosslinks in vivo. The results of these studies will identify the pathways operating in the repair of this medically relevant lesion in vivo and are likely to suggest novel therapeutic approaches that utilize these lesions in the treatment of cancer and other hyperproliferative diseases.