SUMMARY It is intuitively obvious that the ability of a cell to repair DNA damage is saturable, either by limitation of enzymatic activities, the time allotted to achieve their function, or both. However, very little is known regarding the mechanisms that establish such a threshold. Here we demonstrated that the CUL4A ubiquitin ligase restricts the cellular repair capacity by orchestrating the concerted actions of nucleotide excision repair (NER) and the DNA damage-responsive G1/S checkpoint through selective degradation of the DDB2 and XPC DNA damage sensors and the p21/CIP1/WAF1 checkpoint effector. We generated Cul4a conditional knockout mice and observed that skin-specific Cul4a ablation dramatically increased resistance to UV-induced skin carcinogenesis. Our findings reveal that wild-type cells do not operate at their full DNA repair potential, underscore the critical role of CUL4A in establishing the cellular DNA repair threshold, and highlight the potential augmentation of cellular repair proficiency by pharmacological CUL4A inhibition.

DDB1, a component of the Cul4 ubiquitin ligase complex, promotes protein ubiquitination in diverse cellular functions, including nuclear excision repair, regulation of the cell cycle, and DNA replication. To investigate its physiological significance, we generated mice with null and floxed alleles of the DDB1 gene. Here we report that null mutation of DDB1 caused early embryonic lethality, while conditional inactivation of the gene in brain and lens led to neuronal and lens degeneration, brain hemorrhages, and neonatal death. These defects stemmed from a selective elimination of nearly all proliferating neuronal progenitor cells and lens epithelial cells by apoptosis. The cell death was preceded by aberrant accumulation of cell cycle regulators and increased genomic instability and could be partially rescued by removal of the tumor suppressor protein p53. Our results indicate that DDB1 plays an essential role in maintaining viability and genomic integrity of dividing cells.

The HOXA9 homeodomain protein is a key regulator of hematopoiesis and embryonic development. HOXA9 is expressed in primitive hematopoietic cells, and its prompt downregulation is associated with myelocytic maturation. Although transcriptional inactivation of HOXA9 during hematopoietic differentiation has been established, little is known about the biochemical mechanisms underlying the subsequent removal of HOXA9 protein. Here we report that the CUL-4A ubiquitylation machinery controls the stability of HOXA9 by promoting its ubiquitylation and proteasome-dependent degradation. The homeodomain of HOXA9 is responsible for CUL-4A-mediated degradation. Interfering CUL-4A biosynthesis by ectopic expression or by RNA-mediated interference resulted in alterations of the steady-state levels of HOXA9, mirrored by impairment of the ability of 32D myeloid progenitor cells to undergo proper terminal differentiation into granulocytes. These results revealed a novel regulatory mechanism of hematopoiesis by ubiquitindependent proteolysis.

Damaged DNA binding proteins (DDBs) play a critical role in the initial recognition of UV-damaged DNA and mediate recruitment of nucleotide excision repair factors. Previous studies identified DDB2 as a target of the CUL-4A ubiquitin ligase. However, the biochemical mechanism governing DDB proteolysis and its underlying physiological function in the removal of UV-induced DNA damage are largely unknown. Here, we report that the c-Abl nonreceptor tyrosine kinase negatively regulates the repair of UV-induced photolesions on genomic DNA. Biochemical studies revealed that c-Abl promotes CUL-4A-mediated DDB ubiquitination and degradation in a manner that does not require its tyrosine kinase activity both under normal growth conditions and following UV irradiation. Moreover, c-Abl activates DDB degradation in part by alleviating the inhibitory effect of CAND1/TIP120A on CUL-4A. These results revealed a kinase-independent function of c-Abl in a ubiquitin-proteolytic pathway that regulates the damage recognition step of nucleotide excision repair.

The mammalian epidermis is maintained by proliferation and differentiation of epidermal progenitor cells in a stereotyped developmental program. Here we report that tissue-specific deletion of the UV-damaged DNA-binding protein 1 (DDB1) in mouse epidermis led to dramatic accumulation of c-Jun and p21Cip1, arrest of cell cycle at G2/M, selective apoptosis of proliferating cells, and as a result, a nearly complete loss of the epidermis and hair follicles. Deletion of the p53 tumor suppressor gene partially rescued the epithelial progenitor cells from death and allowed for the accumulation of aneuploid cells in the epidermis. Our results suggest that DDB1 plays an important role in development by controlling levels of cell cycle regulators and thereby maintaining genomic stability. The damaged DNA-binding protein complex (DDB), consisting of DDB1 and DDB2, recognizes some UV-damaged DNA lesions and initiates the nucleotide excision repair (NER) process (4). Mutations in DDB2 account for the E group of xeroderma pigmentosum (XP), a repair-deficient disease characterized by a high risk of skin cancer in areas exposed to sunlight (5). Mice with deleted DDB2 exhibit increased skin tumorigenesis after UV-irradiation (6) and develop spontaneous tumors at a high rate when aged (7). DDB1 is evolutionarily conserved from yeast to humans and is likely to play a very fundamental role in cell physiology.Recent work demonstrates that DDB1 functions as an obligatory subunit of the Cullin 4A (Cul4A) E3 ubiquitin ligase and facilitates NER by targeting NER factors such as DDB2 and Cockayne syndrome B protein (CSB) for ubiquitination and degradation (8-10). The DDB1-Cul4A ligase has been shown to target a variety of substrates. These substrates include the DNA replication licensing factor Cdt1 (11, 12) via additional adaptors PCNA (13) and Cdt2 (14, 15), protooncoprotein c-Jun via hDET1 and Cop1 (16), cell-cycle inhibitor p27Kip1 (17), and several histones (18,19), all highlighting the importance of DDB1 in regulating cell cycle and DNA metabolism. Proteomic studies of proteins that interact with DDB1-Cul4A reveal a family of WD40-repeat proteins as substrate-recruiting adaptors for the E3 ligase (15,(20)(21)(22). These adaptors bind to the double ␤-propeller fold of DDB1 and are positioned to present associated substrates to Cul4A, which binds to the third ␤-propeller (20, 23).The myriad substrates of the DDB1-Cul4A ligase suggest that this E3 ligase plays multiple roles beyond NER. In support of this notion, the deletion of DDB1 causes growth defects and changes in nuclear morphology in yeast (24) and lethality early in the development of the fruit fly (25). Recently, we showed that a null mutation of the DDB1 gene in mice leads to early embryonic lethality, and conditional inactivation of the gene in brain and lens eliminates almost all proliferating cells via p53-mediated apoptosis (26). Given the established role of the DDB complex in the NER of UV-damaged DNA lesions in mammalian skin, we attempted to determine the effects of...