ZRF 1 mediates remodeling of E 3 ligases at DNA lesion sites during nucleotide excision repair

Nucleotide excision repair (NER) constitutes one of the major DNA repair pathways. It handles various helix-distorting DNA lesions such as 6–4 photoproducts and cyclobutane pyrimidine dimers (CPDs), arising after exposure to UV light (de Laat et al., 1999). Impaired NER activity is associated with several genetic disorders such as Xeroderma pigmentosum, which is characterized by hypersensitivity to sunlight and a predisposition for skin cancer (Friedberg, 2001). Mammalian NER comprises two pathways that differ in the nature of recognizing DNA lesions. Transcription-coupled (TC) NER is confined to regions of active transcription, where stalled RNA polymerase II triggers the DNA damage response. In contrast, global genome (GG) NER represents the transcription-independent recognition of lesions. The recognition step is followed by verification of the lesion by the repair factor XPA and by the formation of the preexcision complex involving TFI IH and its helicase subunits XPB and XPD. Subsequently, the DNA lesion is excised by the endonucleases XPF and XPG, and the gap is filled by DNA polymerases. (Fousteri and Mullenders, 2008; Marteijn et al., 2014). In GG-NER DNA lesions are recognized by two welldescribed factors: XPC and DDB2. XPC represents a structure specific DNA binding factor, which specifically binds helix-distorting structures (Sugasawa et al., 1998; Riedl et al., 2003). XPC forms a stable complex with the Rad23 homologs RAD23A or RAD23B, respectively, and centrin2 (Masutani et al., 1994; Araki et al., 2001). This trimeric complex binds to a variety of lesions, triggers NER activity, and rapidly dissociates after binding damaged DNA (Sugasawa et al., 2001; Hoogstraten et al., 2008; Bergink et al., 2012). Efficient recognition of CPDs and 6–4 photoproducts also requires the presence of DDB2 (XPE; Tang et al., 2000; Fitch et al., 2003; Moser et al., 2005; Luijsterburg et al., 2007; Nishi et al., 2009). Loss of functional DDB2 causes defective repair of CPDs, reduced repair of 6–4 photoproducts, and hypersensitivity to UV-induced skin cancer (Rapić-Otrin et al., 2003; Alekseev et al., 2005). DDB2 along with DDB1, the RING-domain protein RBX1, and either of the scaffold proteins CUL4A or CUL4B forms E3 ubiquitin ligase complexes (UV–DDB–CUL4A/B) that catalyze the monoubiquitylation of histones H2A, H3, and H4 (Shiyanov et al., 1999; Groisman et al., 2003; Angers et al., 2006; Wang et al., 2006; Guerrero-Santoro et al., 2008). Importantly, the UV–DDB–CUL4A complex catalyzes the polyubiquitylation of XPC, thereby increasing its affinity for DNA in vitro and contributing to recognition and stable binding of photolesions (Sugasawa et al., 2005). A prominent histone modification present at DNA damage sites is ubiquitylation of histones H2A, H2AX, and H1 (Bergink et al., 2006; Mailand et al., 2007; Pan et al., 2011; Thorslund et al., 2015). At double-strand breaks (DSBs), ubiquitylation of Faithful DNA repair is essential to maintain genome integrity. Ultraviolet (UV) irradiation elicits both the recruitment of DNA repair factors and the deposition of histone marks such as monoubiquitylation of histone H2A at lesion sites. Here, we report how a ubiquitin E3 ligase complex specific to DNA repair is remodeled at lesion sites in the global genome nucleotide excision repair (GG-NER) pathway. Monoubiquitylation of histone H2A (H2A-ubiquitin) is catalyzed predominantly by a novel E3 ligase complex consisting of DDB2, DDB1, CUL4B, and RING1B (UV–RING1B complex) that acts early during lesion recognition. The H2A-ubiquitin binding protein ZRF1 mediates remodeling of this E3 ligase complex directly at the DNA lesion site, causing the assembly of the UV–DDB–CUL4A E3 ligase complex (DDB1–DDB2– CUL4A-RBX1). ZRF1 is an essential factor in GG-NER, and its function at damaged chromatin sites is linked to damage recognition factor XPC. Overall, the results shed light on the interplay between epigenetic and DNA repair recognition factors at DNA lesion sites. ZRF1 mediates remodeling of E3 ligases at DNA lesion sites during nucleotide excision repair