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

 描述(申请人提供)：电离辐射(IR)仍然是癌症治疗的支柱之一。IR最有害的损伤是DNA双链断裂(DSB)。双链断裂的准确修复是防止基因组完整性丧失和恶性转化的关键。DSB的有效修复也是许多癌症对放射治疗产生抵抗力的基础。细胞必须选择两条主要的修复途径来修复这些断裂--非同源末端连接(NHEJ)，一种在细胞周期的所有阶段都可操作的容易出错的途径，或者同源重组(HR)，一种被限制在细胞周期的复制后阶段的无错途径。这两条途径的最佳利用对于在基因组侮辱面前保持基因组完整性和细胞生存至关重要。HR的DNA末端切除步骤是正确选择修复途径的关键环节。重要的是，我们实验室和其他实验室的研究已经证实，5‘到3’外切酶EXO1在人类细胞的DNA末端切除和修复途径选择中起关键作用。虽然DNA末端切除目前是DNA修复领域中一个热门的研究课题，但涉及EXO1的分子事件的确切序列并未很好地确定，该序列允许参与特定的修复途径。我们实验室令人振奋的新结果表明，EXO1被CDKs 1/2以细胞周期依赖的方式磷酸化，并被ATM/ATR以DNA损伤依赖的方式磷酸化，从而促进DNA末端切除。然而，在DNA损伤后不久，EXO1被SUMO化、泛素化，并迅速成为降解的目标，推测是为了防止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ExO1‘S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EXO1依赖细胞周期和IR的修饰及其相互作用伙伴，并从机制上了解这些修饰如何促进EXO1的激活和随后对IR的降解。根据我们的初步结果，我们假设EXO1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，这些事件微调DNA末端切除，优化DSB修复，并保持基因组的完整性。了解EXO1翻译后修饰的序列和功能，以及EXO1及其相互作用伙伴在DSB上的准确编排，对于开发针对关键DNA末端切除步骤的更有效的放射增敏方法至关重要。具体地说，我们建议：1)检验CDKs和PI3KKs对EXO1的磷酸化调节DNA末端切除并影响修复途径选择的假设，2)检验辐射后EXO1降解抑制DNA末端切除并保持基因组完整性的假设，3)检验用CDK1/2抑制剂阻断EXO1激活可能是使癌症对电离辐射治疗增敏的可行策略的假设。

 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.