Mechanisms of EXO1 regulation in response to radiation-induced DNA damage

 描述（由申请人提供）：电离辐射（IR）仍然是癌症治疗的主要手段之一。IR引起的最严重的损伤是DNA双链断裂（DSB）。DSB的精确修复对于防止基因组完整性丧失和恶性转化至关重要。DSB的有效修复也是许多癌症对放射疗法的抗性的基础。细胞必须在两种主要的修复途径之间进行选择以修复这些断裂-非同源末端连接（NHEJ），一种在细胞周期的所有阶段中起作用的易错途径，或同源重组（HR），一种限于细胞周期的复制后阶段的无错途径。这两种途径的最佳使用对于在面对基因组损伤时维持基因组完整性和细胞存活至关重要。HR的DNA末端切除步骤是行使正确修复途径选择的关键点。重要的是，我们实验室和其他人的研究已经确定，5'到3'核酸外切酶EXO 1是人类细胞中DNA末端切除和修复途径选择的关键参与者。虽然DNA末端切除目前是DNA修复领域的一个热门研究课题，但涉及EXO 1的分子事件的确切序列并没有很好地确定。来自我们实验室的令人兴奋的新结果表明，EXO 1以细胞周期依赖性方式被CDK 1/2磷酸化，并以DNA损伤依赖性方式被ATM/ATR磷酸化，以促进DNA末端切除。然而，在DNA损伤后不久，EXO 1被SUMO化，泛素化，并迅速降解，可能是为了防止DNA末端的不受控制的切除。重要的是要从机制上理解这些和其他翻译后修饰如何刺激或抑制EXO 1在细胞对IR的反应中的功能。为了实现这一目标，我们建议全面了解细胞周期和IR依赖性修饰以及EXO 1的相互作用伙伴，并从机理上理解这些修饰如何促进EXO 1活化和随后响应IR的降解。基于我们的初步结果，我们假设EXO 1的激活和失活是一个严格控制的过程，涉及磷酸化、SUMO化和泛素化事件，这些事件可以微调DNA末端切除、优化DSB修复和保持基因组完整性。理解EXO 1翻译后修饰的序列和功能以及EXO 1及其相互作用伴侣在DSB处的精确编排对于开发靶向关键DNA末端切除步骤的更有效的放射增敏方法至关重要。具体而言，我们建议：1）检验通过CDK和PI 3KK的EXO 1磷酸化调节DNA末端切除并影响修复途径选择的假设，2）检验辐射后EXO 1降解抑制DNA末端切除并保留基因组完整性的假设，和3）检验用CDK 1/2抑制剂阻断EXO 1活化可能是治疗性地使癌症对电离辐射敏感的可行策略的假设。

 DESCRIPTION (provided by applicant): Ionizing radiation (IR) remains one of the mainstays of cancer therapy. The most deleterious lesion induced by IR is the DNA double-strand break (DSB). Accurate repair of DSBs is essential for preventing loss of genomic integrity and malignant transformation. Efficient repair of DSBs also underlies the resistance of many cancers to radiation therapy. A cell must choose between two major repair pathways to fix these breaks - non- homologous end joining (NHEJ), an error-prone pathway that is operative in all phases of the cell cycle or homologous recombination (HR), an error-free pathway that is restricted to the post-replicative phases of the cell cycle. Optimal usage of these two pathways is vital for the maintenance of genomic integrity and cell survival in the face of genomic insults. The DNA end resection step of HR is a pivotal point at which correct repair pathway choice is exercised. Importantly, research from our lab and others has established that the 5' to 3' exonuclease EXO1 is a critical player in DNA end resection and repair pathway choice in human cells. While DNA end resection is currently an avidly researched topic in the field of DNA repair, the exact sequence of molecular events involving EXO1 that allows commitment to a particular repair pathway is not well worked out. Exciting new results from our lab demonstrate that EXO1 is phosphorylated by CDKs 1/2 in a cell cycle- dependent manner and by ATM/ATR in a DNA damage-dependent manner to promote DNA end resection. However, soon after DNA damage, EXO1 is SUMOylated, ubiquitinated, and rapidly targeted for degradation, presumably to prevent uncontrolled resection of DNA ends. It is important to mechanistically understand how these and other post translational modifications stimulate or restrain EXO1's functions in the cellular response to IR. Towards this goal, we propose to develop a comprehensive picture of cell cycle- and IR-dependent modifications and interacting partners of EXO1, and to mechanistically understand how these modifications promote EXO1 activation and subsequent degradation in response to IR. Based upon our preliminary results, we hypothesize that EXO1 activation and inactivation is a tightly controlled process involving phosphorylation, SUMOylation and ubiquitination events that fine-tune DNA end resection, optimize DSB repair, and preserve genomic integrity. Understanding the sequence and functions of EXO1 post- translational modifications and the exact choreography of EXO1 and its interacting partners at DSBs will be of paramount importance in developing more effective radiosensitization approaches that target the critical DNA end resection step. Specifically, we propose to: 1) Test the hypothesis that phosphorylation of EXO1 by CDKs and PI3KKs regulates DNA end resection and influences repair pathway choice, 2) Test the hypothesis that EXO1 degradation post-radiation restrains DNA end resection and preserves genomic integrity, and 3) Test the hypothesis that blocking EXO1 activation with CDK 1/2 inhibitors may be a viable strategy for therapeutically sensitizing cancers to ionizing radiation.