Coordination of DNA repair and transcription by ubiquitin modification at DNA double strand breaks

摘要 DNA双链断裂(DSB)是最有害的损伤之一。细胞对DSB的反应 涉及复杂的DNA损伤反应网络，用于检测、发出信号并修复损伤；故障 这种系统会导致基因组的突变、缺失和其他变化，并导致许多疾病。邮寄- 通过泛素与蛋白质的共价结合进行的翻译修饰，称为泛素化 在DNA损伤反应中的重要调节作用。之前的研究，包括我们的研究，都表明， 当DSB检测到并激活DNA损伤反应激酶ATM时，Lys63连接的泛素化 受损染色质上的组蛋白H_2A/H_2AX是招募DNA损伤修复蛋白的关键，包括 53BP1和BRCA1，至受损部位。然而，关于泛素的复杂性仍有许多未知之处 修饰及其在DNA损伤反应中的作用。我们已经发现，Lys11-连锁特异性 泛素修饰发生在受损的染色质上，该染色质调节DSB转录的抑制， Lys11-连锁泛素修饰作为一种新的信号和调控平台在应对 DSB。我们进一步证明了这种修饰是ATM依赖的，并由RNF8和UBE2S催化。在……里面 此外，缺乏Lys11连接泛素修饰的细胞表现出对电离的敏感性增加 辐射。这表明DNA修复和转录可以通过不同的连锁特异性来调节 DSB中的泛素修饰。然而，目前尚不清楚这两个看似平行的链接是否特定 泛素化途径被协调来调节细胞反应中的DNA修复和转录 DSB。该建议的目的是确定Lys63和Lys11之间的串扰-连锁修改 研究受损染色质并确定其协调转录的潜在机制 压抑和DNA修复。我们将追求以下具体目标：1)确定 由Cezanne介导的Lys11-和Lys63-连接泛素化调节DNA修复；2)确定其他 协调DNA修复和转录抑制的机制；3)决定 Lys11-和Lys63-连锁泛素化在调节DNA修复和抑制DNA修复中的作用 抄写。我们的发现将为理解泛素修饰是如何在 DSB参与调节转录和修复抑制的信号事件。我们的长期目标是 破译保护基因组完整性的复杂DNA反应网络，这对设计 改善人类健康的工具和治疗。

ABSTRACT DNA double-strand breaks (DSBs) are one of the most deleterious lesions. The cellular responses to DSBs involve a sophisticated DNA damage response network that detects, signals and repairs the lesion; failure of this system results in mutation, deletion and other alteration of genome and underlies many diseases. Post- translational modifications by covalent attachment of ubiquitin to proteins, known as ubiquitination, play important regulatory roles in the DNA damage response. Previous studies, including ours, have illustrated that, upon DSBs detection and activation of the DNA damage response kinase ATM, Lys63-linked ubiquitination of histone H2A/H2AX on damaged chromatin is critical in recruiting DNA damage repair proteins, including 53BP1 and BRCA1, to the damage sites. However, much remains unknown about the complexity of ubiquitin modifications and their roles in the DNA damage response. We have discovered that Lys11-linkage–specific ubiquitin modification occurs on damaged chromatin that regulates repression of transcription at DSBs, revealing Lys11-linkage ubiquitin modification as a new signaling and regulatory platform in the response to DSBs. We further showed that this modification is ATM dependent and catalyzed by RNF8 and Ube2S. In addition, cells deficient in Lys11-linkage ubiquitin modification displayed increased sensitivity to ionizing radiation. It indicates a model that DNA repair and transcription can be regulated by distinct linkage-specific ubiquitin modifications at DSBs. However, it is not clear whether these two seemingly parallel linkage-specific ubiquitination pathways are coordinated to regulate DNA repair and transcription in the cellular responses to DSBs. The objective of this proposal is to identify the crosstalk between Lys63- and Lys11-linkage modification on damaged chromatin and determine the underlying mechanisms through which it coordinates transcriptional repression and DNA repair. We will pursue the following specific aims: 1) determine the crosstalk between Lys11- and Lys63-linkage ubiquitination mediated by Cezanne to regulate DNA repair; 2) determine additional mechanisms underlying the coordination of DNA repair and transcriptional inhibition; 3) determine the role of Lys11- and Lys63-linkage ubiquitination at defined DSB sites in regulating DNA repair and inhibition of transcription. Our findings should provide novel insights into the understanding of how ubiquitin modification at DSBs is involved in signaling events to regulate inhibition of transcription and repair. Our long-term goal is to decipher the complex DNA response network that protects genome integrity, which is essential for design of tools and treatment to improve human health.