Novel regulators of chromatin response to DNA damage

The DNA damage response (DDR) signaling maintains genome stability, thus protecting cells from aging or malignant transformation. DNA damage and following DDR occur in the context of chromatin, and chromatin governs the damage response at multiple levels including post-translational modifications of histone and non-histone proteins. Among these modifications reported at the sites of DNA double-strand breaks (DSBs) are phosphorylation, acetylation, methylation, and ubiquitination. DSB signaling and repair take place in a defined chromatin domain characterized by the enrichment of specific post-translational modifications and accumulated proteins. These structures are microscopically visible and are termed “DNA repair foci". Phosphorylation of serine 139 of histone H2AX (termed γH2AX) is the key event and forms the platform for subsequent repair. The formation and persistence of γH2AX domains reflect chromatin architecture and the efficiency of DSB repair. Another important post-translational modification involved in DSB repair is ubiquitination. Ubiquitination is a covalent protein modification, which requires a three-enzyme cascade. The high abundance of ubiquitin ligases, internal ubiquitin modification sites, and a variety of possible ubiquitin signal structures make ubiquitination one of the most complex modification types in DDR. Due to the complexity large part of the ubiquitin-dependent network remains to be discovered, although several ubiquitin ligases were shown to be involved in the DDR. To identify novel ubiquitin ligases involved in the DDR a human ubiquitinome-wide (663 E3 ubiquitin ligases) high-throughput screening was performed using the formation and persistence of γH2AX foci as a proxy for DSB repair following X-ray exposure. More than 100 novel ubiquitin modifiers that affect DNA damage signaling (30 min post-irradiation) and/or repair (24 h post-irradiation) were identified in the screening. Of the identified hits 62.2% are associated with the damage signaling only (early time point, 107 ...