RESUMO
SLX4, the newly identified Fanconi anemia protein, FANCP, is implicated in repairing DNA damage induced by DNA interstrand cross-linking (ICL) agents, topoisomerase I (TOP1) inhibitors, and in Holliday junction resolution. It interacts with and enhances the activity of XPF-ERCC1, MUS81-EME1, and SLX1 nucleases, but the requirement for the specific nucleases in SLX4 function is unclear. Here, by complementing a null FA-P Fanconi anemia cell line with SLX4 mutants that specifically lack the interaction with each of the nucleases, we show that the SLX4-dependent XPF-ERCC1 activity is essential for ICL repair but is dispensable for repairing TOP1 inhibitor-induced DNA lesions. Conversely, MUS81-SLX4 interaction is critical for resistance to TOP1 inhibitors but is less important for ICL repair. Mutation of SLX4 that abrogates interaction with SLX1 results in partial resistance to both cross-linking agents and TOP1 inhibitors. These results demonstrate that SLX4 modulates multiple DNA repair pathways by regulating appropriate nucleases.
Assuntos
Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA/fisiologia , Anemia de Fanconi/genética , Recombinases/fisiologia , Camptotecina/toxicidade , Linhagem Celular , Reagentes de Ligações Cruzadas/toxicidade , DNA/efeitos dos fármacos , DNA/metabolismo , Análise Mutacional de DNA , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases , Endonucleases/metabolismo , Anemia de Fanconi/enzimologia , Anemia de Fanconi/patologia , Humanos , Mitomicina/toxicidade , Ftalazinas/toxicidade , Piperazinas/toxicidade , Inibidores de Poli(ADP-Ribose) Polimerases , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Recombinases/deficiência , Recombinases/genética , Inibidores da Topoisomerase I/toxicidadeRESUMO
Gain-of-function mutations in the catalytic site of EZH2 (Enhancer of Zeste Homologue 2), is observed in about 22% of diffuse large B-cell lymphoma (DLBCL) cases. Here we show that selective inhibition of histone deacetylase 1,2 (HDAC1,2) activity using a small molecule inhibitor causes cytotoxic or cytostatic effects in EZH2 gain-of-function mutant (EZH2GOF) DLBCL cells. Our results show that blocking the activity of HDAC1,2 increases global H3K27ac without causing a concomitant global decrease in H3K27me3 levels. Our data shows that inhibition of HDAC1,2 is sufficient to decrease H3K27me3 present at DSBs, decrease DSB repair and activate the DNA damage response in these cells. In addition to increased H3K27me3, we found that the EZH2GOF DLBCL cells overexpress another chemotherapy resistance factor - B-lymphoma and BAL-associated protein (BBAP). BBAP monoubiquitinates histone H4K91, a residue that is also subjected to acetylation. Our results show that selective inhibition of HDAC1,2 increases H4K91ac, decreases BBAP-mediated H4K91 monoubiquitination, impairs BBAP-dependent DSB repair and sensitizes the refractory EZH2GOF DLBCL cells to treatment with doxorubicin, a chemotherapy agent. Hence, selective HDAC1,2 inhibition provides a novel DNA repair mechanism-based therapeutic approach as it can overcome both EZH2- and BBAP-mediated DSB repair in the EZH2GOF DLBCL cells.