RESUMO
The DDB1-CUL4-RBX1 (CRL4) ubiquitin ligase family regulates a diverse set of cellular pathways through dedicated substrate receptors (DCAFs). The DCAF DDB2 detects UV-induced pyrimidine dimers in the genome and facilitates nucleotide excision repair. We provide the molecular basis for DDB2 receptor-mediated cyclobutane pyrimidine dimer recognition in chromatin. The structures of the fully assembled DDB1-DDB2-CUL4A/B-RBX1 (CRL4(DDB2)) ligases reveal that the mobility of the ligase arm creates a defined ubiquitination zone around the damage, which precludes direct ligase activation by DNA lesions. Instead, the COP9 signalosome (CSN) mediates the CRL4(DDB2) inhibition in a CSN5 independent, nonenzymatic, fashion. In turn, CSN inhibition is relieved upon DNA damage binding to the DDB2 module within CSN-CRL4(DDB2). The Cockayne syndrome A DCAF complex crystal structure shows that CRL4(DCAF(WD40)) ligases share common architectural features. Our data support a general mechanism of ligase activation, which is induced by CSN displacement from CRL4(DCAF) on substrate binding to the DCAF.
Assuntos
Ubiquitina-Proteína Ligases/química , Animais , Cristalografia por Raios X , Proteínas Culina/química , Dano ao DNA , Proteínas de Ligação a DNA/química , Ativação Enzimática , Humanos , Modelos Moleculares , Ubiquitina-Proteína Ligases/metabolismo , Proteína de Xeroderma Pigmentoso Grupo A/químicaRESUMO
In this Article, in Fig. 1a, the 5' and 3' labels were reversed in the DNA sequence, and Fig. 4 was missing panel labels a-e. These errors have been corrected online.
RESUMO
Access to DNA packaged in nucleosomes is critical for gene regulation, DNA replication and DNA repair. In humans, the UV-damaged DNA-binding protein (UV-DDB) complex detects UV-light-induced pyrimidine dimers throughout the genome; however, it remains unknown how these lesions are recognized in chromatin, in which nucleosomes restrict access to DNA. Here we report cryo-electron microscopy structures of UV-DDB bound to nucleosomes bearing a 6-4 pyrimidine-pyrimidone dimer or a DNA-damage mimic in various positions. We find that UV-DDB binds UV-damaged nucleosomes at lesions located in the solvent-facing minor groove without affecting the overall nucleosome architecture. In the case of buried lesions that face the histone core, UV-DDB changes the predominant translational register of the nucleosome and selectively binds the lesion in an accessible, exposed position. Our findings explain how UV-DDB detects occluded lesions in strongly positioned nucleosomes, and identify slide-assisted site exposure as a mechanism by which high-affinity DNA-binding proteins can access otherwise occluded sites in nucleosomal DNA.
Assuntos
Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , DNA/ultraestrutura , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Dímeros de Pirimidina/análise , Microscopia Crioeletrônica , DNA/química , DNA/efeitos da radiação , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/ultraestrutura , Histonas/química , Histonas/metabolismo , Histonas/ultraestrutura , Humanos , Modelos Moleculares , Nucleossomos/genética , Nucleossomos/efeitos da radiação , Dímeros de Pirimidina/química , Dímeros de Pirimidina/genética , Termodinâmica , Raios Ultravioleta/efeitos adversosRESUMO
Chromatin is the fundamental structure of genomic DNA in eukaryotic cells. The nucleosome, the primary unit of chromatin, consists of DNA and histone proteins, and is important for the maintenance of genomic DNA. Histone mutations are present in many types of cancers, suggesting that chromatin and/or nucleosome structures could be closely related to cancer development. Histone modifications and histone variants are also involved in regulating chromatin and nucleosome structures. Chromatin structures are dynamically changed by nucleosome binding proteins. In this review article, we discuss the current progress toward understanding the relationship between chromatin structure and cancer development.
Assuntos
Histonas , Nucleossomos , Humanos , Histonas/genética , Histonas/metabolismo , Cromatina/genética , DNA/química , Carcinogênese/genéticaRESUMO
The cullin-RING ubiquitin E3 ligase (CRL) family comprises over 200 members in humans. The COP9 signalosome complex (CSN) regulates CRLs by removing their ubiquitin-like activator NEDD8. The CUL4A-RBX1-DDB1-DDB2 complex (CRL4A(DDB2)) monitors the genome for ultraviolet-light-induced DNA damage. CRL4A(DBB2) is inactive in the absence of damaged DNA and requires CSN to regulate the repair process. The structural basis of CSN binding to CRL4A(DDB2) and the principles of CSN activation are poorly understood. Here we present cryo-electron microscopy structures for CSN in complex with neddylated CRL4A ligases to 6.4 Å resolution. The CSN conformers defined by cryo-electron microscopy and a novel apo-CSN crystal structure indicate an induced-fit mechanism that drives CSN activation by neddylated CRLs. We find that CSN and a substrate cannot bind simultaneously to CRL4A, favouring a deneddylated, inactive state for substrate-free CRL4 complexes. These architectural and regulatory principles appear conserved across CRL families, allowing global regulation by CSN.
Assuntos
Biocatálise , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Peptídeo Hidrolases/metabolismo , Peptídeo Hidrolases/ultraestrutura , Regulação Alostérica , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestrutura , Sítios de Ligação , Complexo do Signalossomo COP9 , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Transporte/ultraestrutura , Microscopia Crioeletrônica , Cristalografia por Raios X , Proteínas Culina/química , Proteínas Culina/metabolismo , Proteínas Culina/ultraestrutura , Dano ao DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , Humanos , Cinética , Modelos Moleculares , Complexos Multiproteicos/química , Peptídeo Hidrolases/química , Ligação Proteica , Ubiquitinação , Ubiquitinas/metabolismoRESUMO
In mammalian nucleotide excision repair, the DDB1-DDB2 complex recognizes UV-induced DNA photolesions and facilitates recruitment of the XPC complex. Upon binding to damaged DNA, the Cullin 4 ubiquitin ligase associated with DDB1-DDB2 is activated and ubiquitinates DDB2 and XPC. The structurally disordered N-terminal tail of DDB2 contains seven lysines identified as major sites for ubiquitination that target the protein for proteasomal degradation; however, the precise biological functions of these modifications remained unknown. By exogenous expression of mutant DDB2 proteins in normal human fibroblasts, here we show that the N-terminal tail of DDB2 is involved in regulation of cellular responses to UV. By striking contrast with behaviors of exogenous DDB2, the endogenous DDB2 protein was stabilized even after UV irradiation as a function of the XPC expression level. Furthermore, XPC competitively suppressed ubiquitination of DDB2 in vitro, and this effect was significantly promoted by centrin-2, which augments the DNA damage-recognition activity of XPC. Based on these findings, we propose that in cells exposed to UV, DDB2 is protected by XPC from ubiquitination and degradation in a stochastic manner; thus XPC allows DDB2 to initiate multiple rounds of repair events, thereby contributing to the persistence of cellular DNA repair capacity.
Assuntos
Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Linhagem Celular , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Humanos , Ligação Proteica , Ubiquitinação , Raios UltravioletaRESUMO
The cyclobutane pyrimidine dimer (CPD) is induced in genomic DNA by ultraviolet (UV) light. In mammals, this photolesion is primarily induced within nucleosomal DNA, and repaired exclusively by the nucleotide excision repair (NER) pathway. However, the mechanism by which the CPD is accommodated within the nucleosome has remained unknown. We now report the crystal structure of a nucleosome containing CPDs. In the nucleosome, the CPD induces only limited local backbone distortion, and the affected bases are accommodated within the duplex. Interestingly, one of the affected thymine bases is located within 3.0 Å from the undamaged complementary adenine base, suggesting the formation of complementary hydrogen bonds in the nucleosome. We also found that UV-DDB, which binds the CPD at the initial stage of the NER pathway, also efficiently binds to the nucleosomal CPD. These results provide important structural and biochemical information for understanding how the CPD is accommodated and recognized in chromatin.
Assuntos
DNA/ultraestrutura , Nucleossomos/efeitos da radiação , Nucleossomos/ultraestrutura , Dímeros de Pirimidina/química , Dímeros de Pirimidina/efeitos da radiação , Raios Ultravioleta , Sítios de Ligação , DNA/química , DNA/efeitos da radiação , Ligação de Hidrogênio , Conformação Molecular/efeitos da radiação , Conformação de Ácido Nucleico , Ligação ProteicaRESUMO
Nucleotide excision repair (NER) is a pathway involved in the repair of a variety of potentially mutagenic lesions that distort the DNA double helix. The ubiquitin E3-ligase complex UV-DDB is required for the recognition and repair of UV-induced cyclobutane pyrimidine dimers (CPDs) lesions through NER. DDB2 directly binds CPDs and subsequently undergoes ubiquitination and proteasomal degradation. DDB2 must remain on damaged chromatin, however, for sufficient time to recruit and hand-off lesions to XPC, a factor essential in the assembly of downstream repair components. Here we show that the tumor suppressor USP44 directly deubiquitinates DDB2 to prevent its premature degradation and is selectively required for CPD repair. Cells lacking USP44 have impaired DDB2 accumulation on DNA lesions with subsequent defects in XPC retention. The physiological importance of this mechanism is evident in that mice lacking Usp44 are prone to tumors induced by NER lesions introduced by DMBA or UV light. These data reveal the requirement for highly regulated ubiquitin addition and removal in the recognition and repair of helix-distorting DNA damage and identify another mechanism by which USP44 protects genomic integrity and prevents tumors.
RESUMO
Transcription factors (TFs) regulate gene expression through chromatin where nucleosomes restrict DNA access. To study how TFs bind nucleosome-occupied motifs, we focused on the reprogramming factors OCT4 and SOX2 in mouse embryonic stem cells. We determined TF engagement throughout a nucleosome at base-pair resolution in vitro, enabling structure determination by cryo-electron microscopy at two preferred positions. Depending on motif location, OCT4 and SOX2 differentially distort nucleosomal DNA. At one position, OCT4-SOX2 removes DNA from histone H2A and histone H3; however, at an inverted motif, the TFs only induce local DNA distortions. OCT4 uses one of its two DNA-binding domains to engage DNA in both structures, reading out a partial motif. These findings explain site-specific nucleosome engagement by the pluripotency factors OCT4 and SOX2, and they reveal how TFs distort nucleosomes to access chromatinized motifs.
Assuntos
Regulação da Expressão Gênica , Nucleossomos/química , Fator 3 de Transcrição de Octâmero/química , Fatores de Transcrição SOXB1/química , Animais , Microscopia Crioeletrônica , DNA/química , Histonas/química , Camundongos , Células-Tronco Embrionárias Murinas/metabolismoRESUMO
Huge amounts (>10,000/day) of apurinic/apyrimidinic (AP) sites are produced in genomes, but their structures in chromatin remain undetermined. We determined the crystal structure of the nucleosome containing AP-site analogs at two symmetric sites, which revealed structural polymorphism: one forms an inchworm configuration without an empty space at the AP site, and the other forms a B-form-like structure with an empty space and the orphan base. This unexpected inchworm configuration of the AP site is important to understand the AP DNA repair mechanism, because it may not be recognized by the major AP-binding protein, APE1, during the base excision repair process.
Assuntos
DNA/química , DNA/genética , Conformação de Ácido Nucleico , Nucleossomos/genética , Nucleossomos/metabolismo , Polimorfismo Genético , Polinucleotídeos/química , Dano ao DNA , Reparo do DNA , Histonas/metabolismo , Humanos , Modelos MolecularesRESUMO
The xeroderma pigmentosum group C (XPC) protein complex is a key factor that detects DNA damage and initiates nucleotide excision repair (NER) in mammalian cells. Although biochemical and structural studies have elucidated the interaction of XPC with damaged DNA, the mechanism of its regulation in vivo remains to be understood in more details. Here, we show that the XPC protein undergoes modification by small ubiquitin-related modifier (SUMO) proteins and the lack of this modification compromises the repair of UV-induced DNA photolesions. In the absence of SUMOylation, XPC is normally recruited to the sites with photolesions, but then immobilized profoundly by the UV-damaged DNA-binding protein (UV-DDB) complex. Since the absence of UV-DDB alleviates the NER defect caused by impaired SUMOylation of XPC, we propose that this modification is critical for functional interactions of XPC with UV-DDB, which facilitate the efficient damage handover between the two damage recognition factors and subsequent initiation of NER.
Assuntos
Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/genética , Humanos , Mutação , Ligação Proteica , Proteína SUMO-1/metabolismo , Sumoilação , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação/efeitos da radiação , Raios UltravioletaRESUMO
UV-DDB, an initiation factor for the nucleotide excision repair pathway, recognizes 6-4PP lesions through a base flipping mechanism. As genomic DNA is almost entirely accommodated within nucleosomes, the flipping of the 6-4PP bases is supposed to be extremely difficult if the lesion occurs in a nucleosome, especially on the strand directly contacting the histone surface. Here we report that UV-DDB binds efficiently to nucleosomal 6-4PPs that are rotationally positioned on the solvent accessible or occluded surface. We determined the crystal structures of nucleosomes containing 6-4PPs in these rotational positions, and found that the 6-4PP DNA regions were flexibly disordered, especially in the strand exposed to the solvent. This characteristic of 6-4PP may facilitate UV-DDB binding to the damaged nucleosome. We present the first atomic-resolution pictures of the detrimental DNA cross-links of neighboring pyrimidine bases within the nucleosome, and provide the mechanistic framework for lesion recognition by UV-DDB in chromatin.
Assuntos
DNA/química , Nucleossomos/metabolismo , Dímeros de Pirimidina/química , Raios Ultravioleta , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Histonas/genética , Histonas/metabolismo , Humanos , Maleimidas/química , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Ligação Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
The WD40-repeat protein DDB2 is essential for efficient recognition and subsequent removal of ultraviolet (UV)-induced DNA lesions by nucleotide excision repair (NER). However, how DDB2 promotes NER in chromatin is poorly understood. Here, we identify poly(ADP-ribose) polymerase 1 (PARP1) as a novel DDB2-associated factor. We demonstrate that DDB2 facilitated poly(ADP-ribosyl)ation of UV-damaged chromatin through the activity of PARP1, resulting in the recruitment of the chromatin-remodeling enzyme ALC1. Depletion of ALC1 rendered cells sensitive to UV and impaired repair of UV-induced DNA lesions. Additionally, DDB2 itself was targeted by poly(ADP-ribosyl)ation, resulting in increased protein stability and a prolonged chromatin retention time. Our in vitro and in vivo data support a model in which poly(ADP-ribosyl)ation of DDB2 suppresses DDB2 ubiquitylation and outline a molecular mechanism for PARP1-mediated regulation of NER through DDB2 stabilization and recruitment of the chromatin remodeler ALC1.