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...

The human UV-damaged-DNA binding protein DDB has been linked to the repair deficiency disease xeroderma pigmentosum group E (XP-E), because a subset of XP-E patients lack the damaged-DNA binding function of DDB. Moreover, the microinjection of purified DDB complements the repair deficiency in XP-E cells lacking DDB. Two naturally occurring XP-E mutations of DDB, 82TO and 2RO, have been characterized. They have single amino acid substitutions (K244E and R273H) within the WD motif of the p48 subunit of DDB, and the mutated proteins lack the damaged-DNA binding activity. In this report, we describe a new function of the p48 subunit of DDB, which reveals additional defects in the function of the XP-E mutants. We show that when the subunits of DDB were expressed individually, p48 localized in the nucleus and p125 localized in the cytoplasm. The coexpression of p125 with p48 resulted in an increased accumulation of p125 in the nucleus, indicating that p48 plays a critical role in the nuclear localization of p125. The mutant forms of p48, 2RO and 82TO, are deficient in stimulating the nuclear accumulation of the p125 subunit of DDB. In addition, the mutant 2RO fails to form a stable complex with the p125 subunit of DDB. Our previous studies indicated that DDB can associate with the transcription factor E2F1 and can function as a transcriptional partner of E2F1. Here we show that the two mutants, while they associate with E2F1 as efficiently as wild-type p48, are severely impaired in stimulating E2F1-activated transcription. This is consistent with our observation that both subunits of DDB are required to stimulate E2F1-activated transcription. The results provide insights into the functions of the subunits of DDB and suggest a possible link between the role of DDB in E2F1-activated transcription and the repair deficiency disease XP-E.The human UV-damaged-DNA binding protein has been linked to the repair deficiency disease xeroderma pigmentosum group E (XP-E). Cells from about 30% of XP-E patients (6 of 19) were shown to lack the damaged-DNA binding activity of DDB (3,27). DDB was originally identified as an activity that binds to UV-damaged DNA (3). It has high affinities for the 6-4 photoproduct (14,29,36). In addition, DDB also binds to cisplatin-modified DNA (14). It has been proposed that the damaged-DNA binding activity of DDB is related to a potential DNA repair function (7,15,18,20,36). The microinjection of purified DDB overcomes the repair deficiencies in cells from XP-E patients lacking the damaged-DNA binding activity of DDB (20, 27). However, purified DDB has no significant effect in nucleotide excision repair assays in vitro (18). A recent study proposed that DDB functions as a repair protein in the context of chromatin structure (27). A repair function of DDB would be consistent with the fact that the p48 subunit of DDB possesses extensive sequence homology with the Cockayne syndrome protein CS-A, which is involved in transcription-coupled DNA repair (2,9,12,19).DDB also possesses a transcriptional functi...

The transcription factor E2F1 is believed to be involved in the regulated expression of the DNA replication genes. To gain insights into the transcriptional activation function of E2F1, we looked for proteins in HeLa nuclear extracts that bind to the activation domain of E2F1. Here we show that DDB, a putative DNA repair protein, associates with the activation domain of E2F1. DDB was identified as a heterodimeric protein (48 and 127 kDa) that binds to UV-damaged DNA. We show that the UV-damaged-DNA binding activity from HeLa nuclear extracts can associate with the activation domain of E2F1. Moreover, the 48-kDa subunit of DDB, synthesized in vitro, binds to a fusion protein of E2F1 depending on the C-terminal activation domain. The interaction between DDB and E2F1 can also be detected by coimmunoprecipitation experiments. Immunoprecipitation of an epitope-tagged DDB from cell extracts resulted in the coprecipitation of E2F1. In a reciprocal experiment, immunoprecipitates of E2F1 were found to contain DDB. Fractionation of HeLa nuclear extracts also revealed a significant overlap in the elution profiles of E2F1 and DDB. For instance, DDB, which does not bind to the E2F sites, was enriched in the high-salt fractions containing E2F1 during chromatography through an E2F-specific DNA affinity column. We also observed evidence for a functional interaction between DDB and E2F1 in living cells. For instance, expression of DDB specifically stimulated E2F1-activated transcription. In addition, the transcriptional activation function of a heterologous transcription factor containing the activation domain of E2F1 was stimulated by coexpression of DDB. Moreover, DDB expression could overcome the retinoblastoma protein (Rb)-mediated inhibition of E2F1-activated transcription. The results suggest that this damaged-DNA binding protein can function as a transcriptional partner of E2F1. We speculate that the damaged-DNA binding function of DDB, besides repair, might serve as a negative regulator of E2F1-activated transcription, as damaged DNA will sequester DDB and make it unavailable for E2F1. Furthermore, the binding of DDB to damaged DNA might be involved in downregulating the replication genes during growth arrest induced by damaged DNA.E2F1 is the most-studied member of the E2F family of transcription factors. E2F1 binds the consensus E2F site (TT TCGCGC) as a heterodimer in conjunction with DP1, and it stimulates transcription both in vitro and in vivo (3,13,(20)(21)(22)31). Several genes that are essential for DNA replication and S-phase entry have been shown to be transcriptionally activated by overexpression of E2F1. Included are genes expressing dihydrofolate reductase (4, 26, 56), ribonucleotide reductase, PCNA, DNA polymerase ␣, thymidine kinase, cyclin E, cyclin A, and E2F1, as well as cdc2 (8,42,44). This is consistent with the observation that overexpression of E2F1 induces quiescent cells to enter S phase (2, 30).The activity and levels of E2F1 are regulated very tightly during the progression of the cell cycle. E...

In nonproliferating or growth-arrested cells, the transcription factor E2F remains bound to the retinoblastoma-related protein p130. Accumulation of this E2F-p130 complex correlates with an arrest of the cell cycle progression. Progression through G 1 phase is associated with a cyclin-dependent binding of the cyclindependent kinase cdk2 to the E2F-p130 complex. By fractionating mouse L-cell extracts, we have obtained a partially purified preparation of the E2F-p130 complex that also contains cdk2. Incubation of this complex with recombinant p21 results in a disruption of the interaction between cdk2 and the E2F-p130 complex. We have also analyzed the interaction between cdk2 and the E2F-p130 complex in extracts of a cell line that expresses a temperature-sensitive mutant of p53. Incubation at the permissive temperature (32؇C) results in an induction of p21 synthesis. An increase in the level of p21 in these cells correlates with a loss of cdk2 from the cdk2-containing E2F-p130 complex. We also show that the expression of a reporter gene containing E2F sites in the promoter region is reduced by the coexpression of p21. Since p21 is believed to be a mediator of p53, we speculate that the p21-mediated disruption of the cdk2-containing E2F-p130 complex plays a role in the growth suppression function of p53.The transcription factors of the E2F family play a central role in cell proliferation. Several growth-associated genes, including c-myc, n-myc, B-myb, cdc2, dhfr, and the thymidine synthetase, thymidine kinase, and DNA polymerase ␣ genes, contain E2F-binding sites in their regulatory regions (6,12, 29,33,50,55,56,66,70). Some of these genes have been shown to depend on E2F for their cell cycle-regulated expression (6,12,34,38,43,49,53,64). The activity of E2F is inhibited by proteins of the retinoblastoma family, such as the retinoblastoma tumor suppressor protein (pRB), p107, and p130. Inhibition of E2F activity coincides with a suppression of cell growth by these retinoblastoma family proteins (2,3,4,5,7,8,10,11,14,23,31,32,44,45,57,59,63,67,68,71,73,75).The tumor suppression function of pRB also appears to correlate with its ability to repress E2F-dependent transcription. The naturally occurring loss-of-function mutants of pRB are unable to regulate E2F (2,31,32,57). Recent studies indicated that pRB, p107, and p130 suppress cell growth by biochemically distinct pathways involving different members of the E2F family. pRB regulates E2F1, E2F2, and E2F3, whereas p107 and p130 regulate E2F4 and E2F5 (17, 20, 27, 28, 39, 42, 46, 59, 61, 67, 73). The E2F-regulatory function and growth suppression function of pRB are inactivated by cyclincyclin-dependent kinase (cdk)-dependent phosphorylation of pRB (8,9,13,16,36). p107 and p130 also associate with cyclin-cdk2 complexes (7,11,14,19,24,45,47,48,63,73). Recent experiments indicated that phosphorylation of p107 by a cyclin-cdk complex could regulate its interaction with E2F (74). p107 has been shown to interact with cyclin A-cdk2 through sequences that are distinct fro...

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