Functional regulation of the DNA damage-recognition factor DDB2 by ubiquitination and interaction with xeroderma pigmentosum group C protein

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 capacit

SUMOylation of xeroderma pigmentosum group C protein regulates DNA damage recognition during nucleotide excision repair

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

PARP1 promotes nucleotide excision repair through DDB2 stabilization and recruitment of ALC1

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

The Molecular Basis of CRL4DDB2/CSA Ubiquitin Ligase Architecture, Targeting, and Activation

The DDB1-CUL4-RBX1 (CRL4) ubiquitin ligase family regulates a diverse set of cellular pathways through dedicated substrate receptors (DCAFs). The DCAFDDB2 detects UV-induced pyrimidine dimers in the genome and facilitates nucleotide excision repair. We provide the molecular basis for DDB2 receptormediated cyclobutane pyrimidine dimer recognition in chromatin. The structures of the fully assembled DDB1-DDB2-CUL4A/B-RBX1 (CRL4DDB2) 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 CRL4DDB2 inhibition in a CSN5 independent, nonenzymatic, fashion. In turn, CSN inhibition is relieved upon DNA damage binding to the DDB2 module within CSN-CRL4DDB2. The Cockayne syndrome A DCAF complex crystal structure shows that CRL4DCAF(WD40) ligases share common architectural features. Our data support a general mechanism of ligase activation, which is induced by CSN displacement from CRL4DCAF on substrate binding to the DCAF

Functional regulation of the DNA damage-recognition factor DDB2 by ubiquitination and interaction with xeroderma pigmentosum group C protein

In mammalian nucleotide excision repair, the DDB1–DDB2 complex recognizes UV-induced DNA photolesionsand facilitates recruitment of the XPC complex.Upon binding to damaged DNA, the Cullin 4ubiquitin ligase associated with DDB1–DDB2 is activatedand ubiquitinates DDB2 and XPC. The structurallydisordered N-terminal tail of DDB2 containsseven lysines identified as major sites for ubiquitinationthat target the protein for proteasomal degradation;however, the precise biological functions ofthese modifications remained unknown. By exogenousexpression of mutant DDB2 proteins in normalhuman fibroblasts, here we show that the N-terminaltail of DDB2 is involved in regulation of cellular responsesto UV. By striking contrast with behaviorsof exogenous DDB2, the endogenous DDB2 proteinwas stabilized even after UV irradiation as a functionof the XPC expression level. Furthermore, XPC competitivelysuppressed ubiquitination of DDB2in vitro,and this effect was significantly promoted by centrin-2, which augments the DNA damage-recognition activityof XPC. Based on these findings, we proposethat in cells exposed to UV, DDB2 is protected by XPCfrom ubiquitination and degradation in a stochasticmanner; thus XPC allows DDB2 to initiate multiplerounds of repair events, thereby contributing to thepersistence of cellular DNA repair capacity

Cullin-RING ubiquitin E3 ligase regulation by the COP9 signalosome

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 (CRL4ADDB2) monitors the genome for ultraviolet-light-induced DNA damage. CRL4ADBB2 is inactive in the absence of damaged DNA and requires CSN to regulate the repair process. The structural basis of CSN binding to CRL4ADDB2 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