RESUMO
Repair of DNA alkylation damage is critical for genomic stability and involves multiple conserved enzymatic pathways. Alkylation damage resistance, which is critical in cancer chemotherapy, depends on the overexpression of alkylation repair proteins. However, the mechanisms responsible for this upregulation are unknown. Here, we show that an OTU domain deubiquitinase, OTUD4, is a positive regulator of ALKBH2 and ALKBH3, two DNA demethylases critical for alkylation repair. Remarkably, we find that OTUD4 catalytic activity is completely dispensable for this function. Rather, OTUD4 is a scaffold for USP7 and USP9X, two deubiquitinases that act directly on the AlkB proteins. Moreover, we show that loss of OTUD4, USP7, or USP9X in tumor cells makes them significantly more sensitive to alkylating agents. Taken together, this work reveals a novel, noncanonical mechanism by which an OTU family deubiquitinase regulates its substrates, and provides multiple new targets for alkylation chemotherapy sensitization of tumors.
Assuntos
Alquilação/fisiologia , Dano ao DNA/fisiologia , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA/fisiologia , Dioxigenases/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteases Específicas de Ubiquitina/metabolismo , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato , Alquilação/genética , Western Blotting , Dano ao DNA/genética , Reparo do DNA/genética , Células HEK293 , Humanos , Imunoprecipitação , Microscopia de Fluorescência , Modelos Biológicos , Espectrometria de Massas em TandemRESUMO
53BP1 is a chromatin-associated protein that regulates the DNA damage response. In this study, we identify the TPX2/Aurora A heterodimer, nominally considered a mitotic kinase complex, as a novel binding partner of 53BP1. We find that TPX2/Aurora A plays a previously unrecognized role in DNA damage repair and replication fork stability by counteracting 53BP1 function. Loss of TPX2 or Aurora A compromises DNA end resection, BRCA1 and Rad51 recruitment, and homologous recombination. Furthermore, loss of TPX2 or Aurora A causes deprotection of stalled replication forks upon replication stress induction. This fork protection pathway counteracts MRE11 nuclease activity but functions in parallel to BRCA1. Strikingly, concurrent loss of 53BP1 rescues not only BRCA1/Rad51 recruitment but also the fork instability induced upon TPX2 loss. Our work suggests the presence of a feedback mechanism by which 53BP1 is regulated by a novel binding partner and uncovers a unique role for 53BP1 in replication fork stability.
Assuntos
Aurora Quinase A/metabolismo , Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Recombinação Homóloga , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Animais , Aurora Quinase A/genética , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular/genética , Células HeLa , Humanos , Proteína Homóloga a MRE11/genética , Proteína Homóloga a MRE11/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genéticaRESUMO
The cellular response to DNA double-stranded breaks (DSBs) involves a conserved mechanism of recruitment and activation of numerous proteins involved in this pathway. The events that trigger this response in mammalian cells involve several post-translational modifications, but the role of non-proteasomal ubiquitin signaling is particularly central to this pathway. Recent work has demonstrated that ubiquitination does not act alone, but in concert with other post-translational modifications, including phosphorylation, methylation, acetylation, ADP-ribosylation, and other ubiquitin-like modifiers, particularly SUMOylation. We review novel and exciting crosstalk mechanisms between ubiquitination and other post-translational modifications, many of which work synergistically with each other to activate signaling events and help recruit important DNA damage effector proteins, particularly BRCA1 (breast cancer 1, early onset) and 53BP1 (tumor protein p53 binding protein 1), to sites of DNA damage.
Assuntos
Cromatina/genética , Cromatina/metabolismo , Reparo do DNA/genética , Processamento de Proteína Pós-Traducional/genética , Ubiquitina/genética , Ubiquitina/metabolismo , Animais , Quebras de DNA de Cadeia Dupla , Humanos , Transdução de Sinais/genéticaRESUMO
Histone demethylation is known to regulate transcription, but its role in other processes is largely unknown. We report a role for the histone demethylase LSD1/KDM1A in the DNA damage response (DDR). We show that LSD1 is recruited directly to sites of DNA damage. H3K4 dimethylation, a major substrate for LSD1, is reduced at sites of DNA damage in an LSD1-dependent manner. The E3 ubiquitin ligase RNF168 physically interacts with LSD1 and we find this interaction to be important for LSD1 recruitment to DNA damage sites. Although loss of LSD1 did not affect the initial formation of pH2A.X foci, 53BP1 and BRCA1 complex recruitment were reduced upon LSD1 knockdown. Mechanistically, this was likely a result of compromised histone ubiquitylation preferentially in late S/G2. Consistent with a role in the DDR, knockdown of LSD1 resulted in moderate hypersensitivity to γ-irradiation and increased homologous recombination. Our findings uncover a direct role for LSD1 in the DDR and place LSD1 downstream of RNF168 in the DDR pathway.