Cullin 4A Associates with the UV-damaged DNA-binding Protein DDB

Abstract

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...

Tumor-prone phenotype of the DDB2-deficient mice

“…Interestingly, it was shown that the DDB1/ DDB2/cullin 4A complex remains associated with the Cop9/signalosome (Groisman et al, 2003). UV irradiation causes a dissociation of the interaction with the signalosome and the released DDB1/DDB2/cullin 4A complex associates with the UV-damaged chromatin (Groisman et al, 2003), which is consistent with our previous observation that the DDB1/DDB2/cullin 4A complex binds to UV-damaged DNA with a high affinity (Shiyanov et al, 1999). Therefore, it is possible that DDB2 participates in global nucleotide excision repair by recruiting ubiquitinating enzymes, such as cullin 4A.…”

“…It was shown that DDB2 could be ubiquitinated by cullin 4A (Chen et al, 2001;Nag et al, 2001a). However, the DDB2-DDB1-cullin 4A complex is quite stable inside the cell, as a stable complex can be easily detected in the cell extracts (Shiyanov et al, 1999). Therefore, it is possible that DDB2 functions as a substrate adaptor for ubiquitination.…”

“…Moreover, overexpression of cullin 4A induces ubiquitination and increased decay of DDB2 (Chen et al, 2001;Nag et al, 2001a). However, the proteolysis of DDB2 may not be the only consequence of the DDB2/cullin 4A interaction because a stable complex of DDB1/DDB2/cullin 4A can be easily detected in cell extracts (Shiyanov et al, 1999;Groisman et al, 2003). Interestingly, it was shown that the DDB1/ DDB2/cullin 4A complex remains associated with the Cop9/signalosome (Groisman et al, 2003).…”

Nedd8 on cullin: building an expressway to protein destruction

“…d (Singer et al, 1999;Zhou et al, 2001;Kurz et al, 2002;Ou et al, 2002;Furukawa et al, 2003;Geyer et al, 2003;Pintard et al, 2003b;Xu et al, 2003). e (Shiyanov et al, 1999;Chen et al, 2001;Li et al, 2002;Groisman et al, 2003;Liu et al, 2003;Zhang et al, 2003;Zhong et al, 2003; RNR ¼ ribonucleotide reductase). f (Burnatowska-Hledin et al, 2000;Kamura et al, 2001;Querido et al, 2001).…”

Dynamic two-stage mechanism of versatile DNA damage recognition by xeroderma pigmentosum group C protein

“…A recent crystallographic analysis demonstrated that the binding of UV-DDB to UV lesions is entirely mediated by the DDB2 subunit, which accommodates the crosslinked pyrimidines into a specialized binding pocket and inserts a three-amino acid hairpin into the DNA minor groove [62]. DDB1, on the other hand, is an adaptor that connects the Cul4A-RBX1 ubiquitin ligase 15 to WD40-repeat target proteins [63,64], including DDB2 itself [65,66]. Other known substrates of the Cul4A-RBX1-DDB1 ubiquitin ligase include XPC [67] as well as the core histones H2A, H3 and H4 [68,69].…”