RESUMO
The original version of this Article contained an error in the author affiliations. Xiaochun Yu was incorrectly associated with College of Life Sciences, Hebei University, Baoding 071000 Hebei, China.This has now been corrected in both the PDF and HTML versions of the Article.
RESUMO
53BP1 performs essential functions in DNA double-strand break (DSB) repair and it was recently reported that Tudor interacting repair regulator (TIRR) negatively regulates 53BP1 during DSB repair. Here, we present the crystal structure of the 53BP1 tandem Tudor domain (TTD) in complex with TIRR. Our results show that three loops from TIRR interact with 53BP1 TTD and mask the methylated lysine-binding pocket in TTD. Thus, TIRR competes with histone H4K20 methylation for 53BP1 binding. We map key interaction residues in 53BP1 TTD and TIRR, whose mutation abolishes complex formation. Moreover, TIRR suppresses the relocation of 53BP1 to DNA lesions and 53BP1-dependent DNA damage repair. Finally, despite the high-sequence homology between TIRR and NUDT16, NUDT16 does not directly interact with 53BP1 due to the absence of key residues required for binding. Taken together, our study provides insights into the molecular mechanism underlying TIRR-mediated suppression of 53BP1-dependent DNA damage repair.
Assuntos
Proteínas de Transporte/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ligação Competitiva , Proteínas de Transporte/química , Proteínas de Transporte/genética , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Dano ao DNA , Células HEK293 , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilação , Mutação , Ligação Proteica , Proteínas de Ligação a RNA , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/química , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genéticaRESUMO
DNA double-strand breaks (DSBs) are fatal DNA lesions and activate a rapid DNA damage response. However, the earliest stage of DSB sensing remains elusive. Here, we report that PARP1 and the Ku70/80 complex localize to DNA lesions considerably earlier than other DSB sensors. Using super-resolved fluorescent particle tracking, we further examine the relocation kinetics of PARP1 and the Ku70/80 complex to a single DSB, and find that PARP1 and the Ku70/80 complex are recruited to the DSB almost at the same time. Notably, only the Ku70/80 complex occupies the DSB exclusively in the G1 phase; whereas PARP1 competes with the Ku70/80 complex at the DSB in the S/G2 phase. Moreover, in the S/G2 phase, PARP1 removes the Ku70/80 complex through its enzymatic activity, which is further confirmed by in vitro DSB-binding assays. Taken together, our results reveal PARP1 and the Ku70/80 complex as critical DSB sensors, and suggest that PARP1 may function as an important regulator of the Ku70/80 complex at the DSBs in the S/G2 phase.
Assuntos
Quebras de DNA de Cadeia Dupla , Autoantígeno Ku/genética , Imagem Óptica/métodos , Poli(ADP-Ribose) Polimerase-1/genética , Animais , Núcleo Celular/genética , Dano ao DNA/genética , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/genética , Genoma , Cinética , Autoantígeno Ku/química , Camundongos , Células NIH 3T3 , Poli(ADP-Ribose) Polimerase-1/químicaRESUMO
Poly ADP-ribose polymerases (PARPs) catalyze massive protein poly ADP-ribosylation (PARylation) within seconds after the induction of DNA single- or double-strand breaks. PARylation occurs at or near the sites of DNA damage and promotes the recruitment of DNA repair factors via their poly ADP-ribose (PAR) binding domains. Several novel PAR-binding domains have been recently identified. Here, we summarize these and other recent findings suggesting that PARylation may be the critical event that mediates the first wave of the DNA damage response. We also discuss the potential for functional crosstalk with other DNA damage-induced post-translational modifications.
Assuntos
Dano ao DNA , Reparo do DNA , DNA/genética , Poli Adenosina Difosfato Ribose/metabolismo , Sítios de Ligação/genética , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Quebras de DNA de Cadeia Simples , Humanos , Modelos Biológicos , Fosforilação , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , UbiquitinaçãoRESUMO
OBJECTIVES: A region in the conserved 5' long terminal repeat (LTR) promoter of the integrated HIV-1C provirus was identified for effective targeting by a short double-stranded RNA (dsRNA) to cause heterochromatization leading to a long-lasting decrease in viral transcription, replication and subsequent productive infection in human host cells. METHODS: Small interfering RNAs (siRNAs) were transfected into siHa cells containing integrated LTR-luciferase reporter constructs and screened for efficiency of inducing transcriptional gene silencing (TGS). TGS was assessed by a dual luciferase assay and real-time PCR. Chromatin modification at the targeted region was also studied. The efficacy of potent siRNA was then checked for effectiveness in TZM-bl cells and human peripheral blood mononuclear cells (PBMCs) infected with HIV-1C virus. Viral Gag-p24 antigen levels were determined by ELISA. RESULTS: One HIV-1C LTR-specific siRNA significantly decreased luciferase activity and its mRNA expression with no such effect on HIV-1B LTR. This siRNA-mediated TGS was induced by histone methylation, which leads to heterochromatization of the targeted LTR region. The same siRNA also substantially suppressed viral replication in TZM-bl cells and human PBMCs infected with various HIV-1C clinical isolates for ≥3 weeks after a single transfection, even of a strain that had a mismatch in the target region. CONCLUSIONS: We have identified a potent dsRNA that causes long-term suppression of HIV-1C virus production in vitro and ex vivo by heritable epigenetic modification at the targeted C-LTR region. This dsRNA has promising therapeutic potential in HIV-1C infection, the clade responsible for more than half of AIDS cases worldwide.
