RESUMEN
The chromatin remodeler ALC1 is recruited to and activated by DNA damage-induced poly(ADP-ribose) (PAR) chains deposited by PARP1/PARP2/HPF1 upon detection of DNA lesions. ALC1 has emerged as a candidate drug target for cancer therapy as its loss confers synthetic lethality in homologous recombination-deficient cells. However, structure-based drug design and molecular analysis of ALC1 have been hindered by the requirement for PARylation and the highly heterogeneous nature of this post-translational modification. Here, we reconstituted an ALC1 and PARylated nucleosome complex modified in vitro using PARP2 and HPF1. This complex was amenable to cryo-EM structure determination without cross-linking, which enabled visualization of several intermediate states of ALC1 from the recognition of the PARylated nucleosome to the tight binding and activation of the remodeler. Functional biochemical assays with PARylated nucleosomes highlight the importance of nucleosomal epitopes for productive remodeling and suggest that ALC1 preferentially slides nucleosomes away from DNA breaks.
Asunto(s)
Proteínas Portadoras/metabolismo , Ensamble y Desensamble de Cromatina , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Nucleosomas/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli ADP Ribosilación , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Portadoras/genética , Microscopía por Crioelectrón , ADN Helicasas/genética , ADN Helicasas/ultraestructura , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/ultraestructura , Humanos , Cinética , Modelos Moleculares , Proteínas Nucleares/genética , Nucleosomas/genética , Nucleosomas/ultraestructura , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasas/genética , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
KDM4D is a lysine demethylase that removes tri- and di- methylated residues from H3K9 and is involved in transcriptional regulation and carcinogenesis. We recently showed that KDM4D is recruited to DNA damage sites in a PARP1-dependent manner and facilitates double-strand break repair in human cells. Moreover, we demonstrated that KDM4D is an RNA binding protein and mapped its RNA-binding motifs. Interestingly, KDM4D-RNA interaction is essential for its localization on chromatin and subsequently for efficient demethylation of its histone substrate H3K9me3. Here, we provide new data that shed mechanistic insights into KDM4D accumulation at DNA damage sites. We show for the first time that KDM4D binds poly(ADP-ribose) (PAR) in vitro via its C-terminal region. In addition, we demonstrate that KDM4D-RNA interaction is required for KDM4D accumulation at DNA breakage sites. Finally, we discuss the recruitment mode and the biological functions of additional lysine demethylases including KDM4B, KDM5B, JMJD1C, and LSD1 in DNA damage response.
Asunto(s)
Daño del ADN , Histona Demetilasas con Dominio de Jumonji/metabolismo , Sitios de Unión , Línea Celular Tumoral , Reparación del ADN , Inestabilidad Genómica , Humanos , Unión Proteica , Transporte de Proteínas , ARN no Traducido/metabolismoRESUMEN
The posttranslational modification of proteins with poly(ADP-ribose) (PAR) regulates protein-protein interactions in DNA repair, gene expression, chromatin structure, and cell fate determination. The PAR polymerase PARP1 binds to damaged chromatin and synthesizes PAR chains to signal DNA damage and recruit the DNA repair scaffold, XRCC1. Pharmacological blockade of PARP1 enzymatic activity impairs XRCC1-dependent repair of DNA damage and selectively kills cancer cells lacking other DNA repair functions. As such, PARP inhibitors are promising new therapies for repair-deficient tumors such as BRCA mutated breast cancers. Although the XRCC1-PARP1 complex is relevant to the proposed therapeutic mechanism of PARP inhibitors, the physical makeup and dynamics of this complex are not well characterized at the molecular level. Here we describe a fluorescence-based, real-time assay that quantitatively monitors interactions between PARylated PARP1 and XRCC1. Using this assay, we show that the PAR posttranslational modification by itself is a high affinity ligand for XRCC1, requiring a minimum chain length of 7 ADP-ribose units in the oligo(ADP-ribose) ligand for a stable interaction with XRCC1. This discrete binding interface enables the PAR glycohydrolase (PARG) to completely disassemble the PARP1-XRCC1 complex without assistance from a mono(ADP-ribose) glycohydrolase. Our quantitative, real-time assay of PAR-dependent protein-protein interactions and PAR turnover by PARG is an excellent tool for high-throughput screening to identify pharmacological modulators of PAR metabolism that may be useful therapeutic alternatives to PARP inhibitors.