Project 2: Error-free and Mutagenic Processing of Crosslinks

项目总结 DNA链间交联(ICL)是DNA代谢过程的强大障碍，必须修复 为了细胞的生存。由于ICL的毒性很大，许多ICL诱导剂被用作抗癌治疗药物。 这类化疗药物的使用也会导致DNA-蛋白质交联物(DPC)的形成。 虽然来自几个修复途径的蛋白质被认为参与了ICL和DPC的处理，但 哺乳动物细胞的ICL和DPC修复机制尚不清楚。因此，该委员会的总体目标是 建议的研究是通过阐明交联链修复的机制来填补这一知识空白 哺乳动物细胞。我们和其他人已经证明，来自几个修复途径的蛋白质参与了 ICL修复，包括核苷酸切除修复(NER)、错配修复(MMR)、同源重组 (HR)、Fanconi贫血(FA)和高迁移率族(HMGB)蛋白。然而，它们的功能和 哺乳动物细胞ICL修复中的相互作用尚不清楚。需要检验的新假说包括 ERCC1-XPF以SLX4或uhrf1依赖的方式参与ICL的初始解钩； HMGB1，而不是HMGB3，参与ERCC1-XPF到ICL的招募，以促进在 SLX4不依赖方式；ICL诱导的DSB由诱变的和无错误的HR处理 哺乳动物细胞中受HMGB蛋白特异性调控的通路。长期目标是 阐明与去除哺乳动物基因组中的交联链有关的分子机制。 具体地说，我们建议：(A)在体内确定交联过程中的切割和诱变机制 修复；(B)阐明与ICL相关的无错误和诱变的交联链修复所涉及的机制 诱导DSB的形成；以及(C)确定DPC在人类细胞中的突变和重组影响。 拟议的工作具有创新性，因为它将检验新的假设；此外， 方法是在哺乳动物基因组中诱导位点特异性ICL来研究它们的修复。此外，从基因上讲， 为这个项目构建的工程哺乳动物细胞系将允许最终评估 修复体内ICL加工中的蛋白质，方法是将最初的解钩步骤与随后的DSB- 诱导HR加工。这些实验将定义新组件的功能(例如，uhrf1和 HMGB蛋白)参与ICL修复，并有可能识别尚未确定的 参与交联链修复的蛋白质/途径。预期的贡献是ICL的机制 而哺乳动物细胞中的DPC修复将被阐明，这一点具有重要意义，因为新的信息 所获得的结果将有助于开发利用交联剂控制人类癌症的新的靶向策略 探员们。这些研究还将为遗传学领域贡献重要的基本新知识 不稳定和DNA损伤与修复。

PROJECT SUMMARY DNA interstrand crosslinks (ICLs) present formidable blocks to DNA metabolic processes and must be repaired for cell survival. Because ICLs are so toxic, many ICL-inducing agents are used as anticancer therapeutics. The use of such chemotherapeutic agents can also result in the formation of DNA-protein crosslinks (DPCs). While proteins from several repair pathways are thought to be involved in processing ICLs and DPCs, the mechanisms of ICL and DPC repair in mammalian cells are not clearly defined. Thus, the overall goal of the proposed studies is to fill this gap in knowledge by elucidating the mechanisms of crosslink repair in mammalian cells. We and others have demonstrated that proteins from several repair pathways are involved in ICL repair, including nucleotide excision repair (NER), mismatch repair (MMR), homologous recombination (HR), Fanconi anemia (FA), and high mobility group (HMGB) proteins. However, their functions and interactions in ICL repair in mammalian cells are not well understood. The novel hypotheses to be tested are that: ERCC1-XPF is involved in the initial unhooking of ICLs in an SLX4- or UHRF1-dependent fashion; that HMGB1, but not HMGB3, is involved in the recruitment of ERCC1-XPF to ICLs to facilitate unhooking in an SLX4-independent fashion; and that ICL-induced DSBs are processed by mutagenic and error-free HR pathways, specifically regulated by HMGB proteins in mammalian cells. The long-term objectives are to elucidate molecular mechanisms involved in the removal of crosslinks from the mammalian genome. Specifically we propose to: (a) determine in vivo the mechanisms of incision and mutagenesis during crosslink repair; (b) elucidate the mechanisms involved in error-free and mutagenic crosslink repair associated with ICL- induced DSB formation; and (c) determine the mutagenic and recombinogenic impact of DPCs in human cells. The proposed work is innovative because it will test novel hypotheses; moreover, a unique feature of the approach is to induce site-specific ICLs in mammalian genomes to study their repair. In addition, genetically engineered mammalian cells lines constructed for this project will allow for definitive assessment of the roles of repair proteins in ICL processing in vivo, by separating the initial unhooking steps from the subsequent DSB- induced HR processing. These experiments will define the function of novel components (e.g. UHRF1 and HMGB proteins) involved in ICL repair, and have the potential to identify as yet undetermined proteins/pathways involved in the repair of crosslinks. The expected contribution is that the mechanisms of ICL and DPC repair in mammalian cells will be elucidated, which is significant because the new information obtained will aid in the development of novel targeted strategies to control human cancers using crosslinking agents. The studies will also contribute significant fundamental new knowledge to the fields of genetic instability and DNA damage and repair.