RESUMO
The WD40 domain-containing protein WRAP53ß (WD40 encoding RNA antisense to p53; also referred to as WDR79/TCAB1) controls trafficking of splicing factors and the telomerase enzyme to Cajal bodies, and its functional loss has been linked to carcinogenesis, premature aging, and neurodegeneration. Here, we identify WRAP53ß as an essential regulator of DNA double-strand break (DSB) repair. WRAP53ß rapidly localizes to DSBs in an ATM-, H2AX-, and MDC1-dependent manner. We show that WRAP53ß targets the E3 ligase RNF8 to DNA lesions by facilitating the interaction between RNF8 and its upstream partner, MDC1, in response to DNA damage. Simultaneous binding of MDC1 and RNF8 to the highly conserved WD40 scaffold domain of WRAP53ß facilitates their interaction and accumulation of RNF8 at DSBs. In this manner, WRAP53ß controls proper ubiquitylation at DNA damage sites and the downstream assembly of 53BP1, BRCA1, and RAD51. Furthermore, we reveal that knockdown of WRAP53ß impairs DSB repair by both homologous recombination (HR) and nonhomologous end-joining (NHEJ), causes accumulation of spontaneous DNA breaks, and delays recovery from radiation-induced cell cycle arrest. Our findings establish WRAP53ß as a novel regulator of DSB repair by providing a scaffold for DNA repair factors.
Assuntos
Reparo do DNA/fisiologia , Telomerase/metabolismo , Ubiquitina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Células Cultivadas , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Histonas/metabolismo , Humanos , Chaperonas Moleculares , Proteínas Nucleares/metabolismo , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Telomerase/genética , Transativadores/metabolismo , Ubiquitina-Proteína LigasesRESUMO
The mechanism of multicellular drug resistance, defined as the reduced efficacy of chemotherapeutic drugs in solid tumors is incompletely understood. Here we report that colon carcinoma cells cultured as 3D microtissues (spheroids) display dramatic increases in the expression of a subset of type I interferon-(IFN)-stimulated genes (ISGs). A similar gene signature was associated previously with resistance to radiation and chemotherapy, prompting us to examine the underlying biological mechanisms. Analysis of spheroids formed by different tumor cell lines and studies using knock-down of gene expression showed that cell crowding leads to the induction of IFN regulatory factor-9 (IRF9) which together with STAT2 and independently of IFNs, is necessary for ISG upregulation. Increased expression of IRF9 alone was sufficient to induce the ISG subset in monolayer cells and to confer increased resistance to clinically used cytotoxic drugs. Our data reveal a novel mechanism of regulation of a subset of ISGs, leading to drug resistance in solid tumors.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Apoptose , Comunicação Celular , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , Interferons/fisiologia , Fator de Transcrição STAT2/metabolismo , Ativação TranscricionalRESUMO
We recently demonstrated that WRAP53ß acts as a key regulator of ubiquitin-dependent repair of DNA double-strand breaks. Here, we applied the proximity ligation assay (PLA) to show that at such breaks WRAP53ß accumulates in close proximity to γH2AX and, furthermore as demonstrated by their co-immunoprecipitation (IP) binds to γH2AX, in a manner dependent on the ATM and ATR kinases. Moreover, formation of complexes between MDC1 and both its partners RNF8 and phosphorylated ATM was visualized. The interaction of MDC1 with RNF8, but not with ATM requires WRAP53ß, suggesting that WRAP53ß facilitates the former interaction without altering phosphorylation of MDC1 by ATM. Furthermore, our findings highlight PLA as a more sensitive method for the analysis of recruitment of repair factors and complex formation at DNA breaks that are difficult to detect using conventional immunofluorescence.