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 涉及一个复杂的DNA损伤反应网络，检测，信号和修复病变; 该系统导致基因组的突变、缺失和其他改变，并成为许多疾病的基础。后 通过泛素与蛋白质的共价连接进行的翻译修饰，称为泛素化， 在DNA损伤反应中的重要调节作用。以前的研究，包括我们的研究，已经表明， 在DSB检测和DNA损伤反应激酶ATM激活后， 受损染色质上的组蛋白H2 A/H2 AX在招募DNA损伤修复蛋白中至关重要，包括 53 BP 1和BRCA 1，到损伤部位。然而，关于泛素的复杂性， 修饰及其在DNA损伤反应中的作用。我们发现Lys 11-linkage特异性 泛素修饰发生在受损的染色质上，其调节DSB处的转录抑制， 揭示了Lys 11-连接泛素修饰作为一种新的信号传导和调节平台， DSB。我们进一步表明，这种修饰是ATM依赖性的，并由RNF 8和Ube 2S催化。在 此外，Lys 11-连接泛素修饰缺陷的细胞显示对电离辐射的敏感性增加， 辐射这表明DNA修复和转录可以通过不同的连接特异性调控的模型。 DSB处的泛素修饰。然而，目前尚不清楚这两个看似平行的联系是否具体 泛素化途径协调调节DNA修复和转录的细胞反应， DSB。本建议的目的是确定Lys 63-和Lys 11-连接修饰之间的串扰 并确定其协调转录的潜在机制 抑制和DNA修复。我们将追求以下具体目标：1）确定 Cezanne介导的Lys 11-和Lys 63-连接泛素化调节DNA修复; 2）确定额外的 DNA修复和转录抑制的协调机制; 3）确定 DSB位点上的Lys 11和Lys 63连接泛素化在调节DNA修复和抑制细胞凋亡中的作用 转录。我们的研究结果应该提供新的见解，了解泛素修饰是如何在 DSB参与信号传导事件以调节转录抑制和修复。我们的长期目标是 破译复杂的DNA反应网络，保护基因组的完整性，这是至关重要的设计， 改善人类健康的工具和治疗方法。

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.