The damaged-DNA binding protein DDB consists of two subunits, DDB1 (127 kDa) and DDB2 (48 kDa). Mutations in the DDB2 subunit have been detected in patients suffering from the repair deficiency disease xeroderma pigmentosum (group E). In addition, recent studies suggested a role for DDB2 in global genomic repair. DDB2 also exhibits transcriptional activity. We showed that expression of DDB1 and DDB2 stimulated the activity of the cell cycle regulatory transcription factor E2F1. Here we show that DDB2 is a cell cycleregulated protein. It is present at a low level in growth-arrested primary fibroblasts, and after release the level peaks at the G 1 /S boundary. The cell cycle regulation of DDB2 involves posttranscriptional mechanisms. Moreover, we find that an inhibitor of 26S proteasome increases the level of DDB2, suggesting that it is regulated by the ubiquitin-proteasome pathway. Our previous study indicated that the cullin family protein Cul-4A associates with the DDB2 subunit. Because cullins are involved in the ubiquitin-proteasome pathway, we investigated the role of Cul-4A in regulating DDB2. Here we show that DDB2 is a specific target of Cul-4A. Coexpression of Cul-4A, but not Cul-1 or other highly related cullins, increases the ubiquitination and the decay rate of DDB2. A naturally occurring mutant of DDB2 (2RO), which does not bind Cul-4A, is not affected by coexpression of Cul-4A. Studies presented here identify a specific function of the Cul-4A gene, which is amplified and overexpressed in breast cancers.The DDB2 subunit (also referred to as p48 or p48DDB) of the damaged-DNA binding protein DDB is mutated in xeroderma pigmentosum (group E, XP-E). Several naturally occurring mutants of DDB2 have been identified and characterized (36,37,41). These mutants are deficient in damaged-DNA recognition (17,36,37). One of these mutants, referred to as 2RO, also fails to associate with the DDB1 subunit (also referred to as p127 or p127DDB) (42); a patient harboring this mutation developed malignant skin cancer and died at the age of 14 (7). Another mutant, 82TO, is able to associate with the DDB1 subunit but is impaired in its ability to stimulate nuclear localization of DDB1 (42). A patient harboring the 82TO mutation exhibited severe sun sensitivity (25). In addition, a nonsense mutation in the DDB2 gene was correlated with the development of multiple skin neoplasia (18a). The incidence of tumors in patients with DDB2 mutations suggests a role for DDB2 in the pathway of tumor suppression. The damaged-DNA binding function of DDB2 suggested a role in DNA repair, which was supported by several lines of evidence. First, fibroblasts, isolated from the XP-E patients harboring mutations in the DDB2 gene, exhibited reduced DNA repair activity (17,21,41). In addition, microinjection of the wild-type protein could complement the deficiency in cells harboring mutant DDB2 (21, 41). Recent studies suggested that DDB is involved in global genomic repair (17, 18, 47). Furthermore, it was shown that DDB2 is downstream of p53 ...

The damaged DNA-binding protein (DDB) is believed to be involved in DNA repair, and it has been linked to the repair deficiency disease xeroderma pigmentosum. DDB also exhibits transcriptional activities. DDB binds to the activation domain of E2F1 and stimulates E2F1-activated transcription. Here we provide evidence that DDB or DDB-associated proteins are targets of cullin 4A (CUL-4A). CUL-4A is a member of the cullin family of proteins, which are believed to be ubiquitin-protein isopeptide ligases (type E3). The CUL-4A gene has been shown to be amplified and up-regulated in breast carcinomas. In this study, we identify CUL-4A as one of the DDB-associated proteins. CUL-4A co-immunoprecipitates with DDB, but not with a naturally occurring mutant of DDB. Moreover, CUL-4A in HeLa nuclear extracts co-purifies with DDB, suggesting they are parts of the same complex. The observation provides insights how CUL-4A, through an interaction with DDB, might be playing a role in the development of breast carcinomas. DDB1 binds to UV-damaged DNA and cisplatin modified DNA with high affinities (1-3). The damaged DNA binding function of DDB requires both DDB1 (p125, 127 kDa) and DDB2 (p48, 48 kDa) gene products, which are believed to be subunits of DDB (2, 4). About 30% of XP-E (xeroderma pigmentosum group E) patients lack the damaged DNA binding activity of DDB (5, 6). DDB exhibits very little repair activity in nucleotide excision repair assays, in vitro (7). However, microinjection of purified DDB complements the repair deficiencies in XP-E cells lacking the damaged-DNA binding activity of DDB (8). It has been postulated that DDB functions as a repair protein in the context of chromatin structure and that it alters chromatin conformation to enhance repair at the damaged sites (4, 6). Recent results also suggest that damaged DNA recognition function of DDB is downstream of p53. Induced expression of p53 causes an increase in the expression of p48 mRNA (9). Moreover, p48 mRNA expression is increased upon DNA damage in p53 ϩ/ϩ cells, but not in p53 Ϫ/Ϫ cells.Two mutants, 2RO and 82TO, have been characterized from XP-E patients. These mutants harbor single amino acid substitutions, R273H (2RO) and K244E (82TO), in the WD motif of p48 (DDB2 gene product) (10). These mutant p48 proteins are impaired in their ability to cooperate with the p125 subunit in damaged DNA binding assays (4, 10). The mutant 2RO is also incapable of forming a stable complex with the p125 subunit (11). p48 plays an important role in the nuclear localization of p125 (11). These two XP-E mutants of p48 are deficient in their ability to enhance nuclear localization of p125 (11).DDB also possesses a transcriptional function (11-13). It can function as a transcriptional partner of E2F1. DDB associates with the C-terminal activation domain of E2F1 and cooperates with E2F1 to stimulate transcription from an E2F1-regulated promoter (13). Moreover, expression of DDB can overcome retinoblastoma inhibition of the E2F1-activated transcription (13). The transcriptional f...

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DDB1, a subunit of the damaged-DNA binding protein DDB, has been shown to function also as an adaptor for Cul4A, a member of the cullin family of E3 ubiquitin ligase. The Cul4A-DDB1 complex remains associated with the COP9 signalosome, and that interaction is conserved from fission yeast to human. Studies with fission yeast suggested a role of the Pcu4-Ddb1-signalosome complex in the proteolysis of the replication inhibitor Spd1. Here we provide evidence that the function of replication inhibitor proteolysis is conserved in the mammalian DDB1-Cul4A-signalosome complex. We show that small interfering RNA-mediated knockdown of DDB1, CSN1 (a subunit of the signalosome), and Cul4A in mammalian cells causes an accumulation of p27 Kip1 . Moreover, expression of DDB1 reduces the level of p27 Kip1 by increasing its decay rate. The DDB1-induced proteolysis of p27Kip1 requires signalosome and Cul4A, because DDB1 failed to increase the decay rate of p27Kip1 in cells deficient in CSN1 or Cul4A. Surprisingly, the DDB1-induced proteolysis of p27 Kip1 also involves Skp2, an F-box protein that allows targeting of p27Kip1 for ubiquitination by the Skp1-Cul1-F-box complex. Moreover, we provide evidence for a physical association between Cul4A, DDB1, and Skp2. We speculate that the F-box protein Skp2, in addition to utilizing Cul1-Skp1, utilizes Cul4A-DDB1 to induce proteolysis of p27 Kip1 .The Cul4A gene is amplified and overexpressed in breast and hepatocellular carcinomas (6, 42). Also, Cul4A is essential for mammalian development (18). It encodes a protein of the cullin family. The cullins are central components of several E3 ubiquitin ligases (11). Cul4A associates with the damaged-DNA binding protein DDB (22,32). DDB consists of two subunits: DDB1 and DDB2. The DDB2 subunit is mutated in xeroderma pigmentosum (complementation group E) (reviewed in reference 35). Cul4A participates in the ubiquitination of the DDB2 subunit of DDB and induces its proteolysis through the ubiquitin-proteasome pathway (22). Recent studies indicated that the DDB1 subunit of DDB functions as an adaptor for substrate binding by Cul4A in a manner similar to how Skp1 functions in the Skp1-cullin1-F-box (SCF) complex (15). However, unlike the case for Skp1, there are instances where DDB1 directly targets a substrate without additional adaptor proteins. For example, Cul4A has been implicated in the proteolysis of the replication licensing protein Cdt1 following DNA damage (14, 44). It was shown that the interaction between Cul4A and Cdt1 is mediated by DDB1 (15). In other examples, Cul4A-DDB1 interacts with additional adaptors to target a specific protein. The DDB1-Cul4A complex associates with hDET1, an ortholog of Arabidopsis De-etiolated-1, and hCOP1, an ortholog of Arabidopsis constitutively photomorphogenic-1 (COP1), to induce proteolysis of the c-Jun protein through the ubiquitin-proteasome pathway (40). In that study, the authors proposed that the hDET1-hCOP1 functioned as the heteromeric substrate adaptor and, in keeping with the SCF E3 ligase,...