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

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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.