Ubiquitin-activating enzyme UBA1 is required for cellular response to DNA damage

Abstract

The cellular DNA damage response (DDR) machinery that maintains genomic integrity and prevents severe pathologies, including cancer, is orchestrated by signaling through protein modifications. Protein ubiquitylation regulates repair of DNA double-strand breaks (DSBs), toxic lesions caused by various metabolic as well as environmental insults such as ionizing radiation (IR). Whereas several components of the DSB-evoked ubiquitylation cascade have been identified, including RNF168 and BRCA1 ubiquitin ligases, whose genetic defects predispose to a syndrome mimicking ataxia-telangiectasia and cancer, respectively, the identity of the apical E1 enzyme involved in DDR has not been established. Here, we identify ubiquitin-activating enzyme UBA1 as the E1 enzyme required for responses to IR and replication stress in human cells. We show that siRNA-mediated knockdown of UBA1, but not of another UBA family member UBA6, impaired formation of both ubiquitin conjugates at the sites of DNA damage and IR-induced foci (IRIF) by the downstream components of the DSB response pathway, 53BP1 and BRCA1. Furthermore, chemical inhibition of UBA1 prevented IRIF formation and severely impaired DSB repair and formation of 53BP1 bodies in G 1, a marker of response to replication stress. In contrast, the upstream steps of DSB response, such as phosphorylation of histone H2AX and recruitment of MDC1, remained unaffected by UBA1 depletion. Overall, our data establish UBA1 as the apical enzyme critical for ubiquitylation-dependent signaling of both DSBs and replication stress in human cells, with implications for maintenance of genomic integrity, disease pathogenesis and cancer treatment.

UbcH7 regulates 53BP1 stability and DSB repair

“…Several studies reported the screening of genes in regulating the recruitment of 53BP1 to DNA damage sites (38)(39)(40). Depletion of UbcH7 was reported to increase the size of 53BP1 foci (30), similar to our observation.…”

Downregulation of Wip1 phosphatase modulates the cellular threshold of DNA damage signaling in mitosis

“…Recent studies identified multiple protein complexes that control DNA-damage induced histone ubiquitination, and these include E3 ligases RNF8, 4,5 RNF168 6 and HERC2; 7 E2-conjugating enzyme Ubc13 and E1 ubiquitin-activating enzyme UBA1. 8 Both histone phosphorylation and ubiquitination are necessary for recruitment of 53BP1 and BRCA1 proteins, essential regulators of non-homologous end joining and homologous recombination DNA repair pathways, respectively. 2,9 Besides the well-established role in DNA repair of lesions caused by ionizing radiation (IR) or by genetic rearrangements during maturation of the immune system, for example, 53BP1 is also involved in protecting endogenous underreplicated DNA regions (such as fragile sites) during interphase.…”

The E3 Ubiquitin Ligase ARIH1 Protects against Genotoxic Stress by Initiating a 4EHP-Mediated mRNA Translation Arrest

“…Moreover, we identified seven siRNAs targeting proteins with no described ubiquitinase function that were included in the ThermoFisher "ubiquitination library," presumably based on the presence of predicted domains associated with ubiquitinase function, including RING, SOCS, or SPRY (see Discussion). The knockdown of the E1 ubiquitin enzyme Ube1x (Uba), which has recently been shown to be a crucial E1 enzyme in the DDR following ionizing radiation and replication stress (29), resulted in a particularly strong reduction of viability (Fig. 1C).…”