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Reactive oxygen species (ROS) is critical for premature senescence, a process significant in tumor suppression and cancer therapy. Here, we reveal a novel function of the nucleotide excision repair protein DDB2 in the accumulation of ROS in a manner that is essential for premature senescence. DDB2-deficient cells fail to undergo premature senescence induced by culture shock, exogenous oxidative stress, oncogenic stress, or DNA damage. These cells do not accumulate ROS following DNA damage. The lack of ROS accumulation in DDB2 deficiency results from high-level expression of the antioxidant genes in vitro and in vivo. DDB2 represses antioxidant genes by recruiting Cul4A and Suv39h and by increasing histone-H3K9 trimethylation. Moreover, expression of DDB2 also is induced by ROS. Together, our results show that, upon oxidative stress, DDB2 functions in a positive feedback loop by repressing the antioxidant genes to cause persistent accumulation of ROS and induce premature senescence.

DDB2 was identified as a protein involved in the Nucleotide Excision Repair (NER), a major DNA repair mechanism that repairs UV damage to prevent accumulation of mutations and tumorigenesis. However, recent studies indicated additional functions of DDB2 in the DNA damage response pathway. Herein, we discuss the proposed mechanisms by which DDB2 activates NER and programmed cell death upon DNA damage through its E3 ligase activity.

The xeroderma pigmentosum group E gene product DDB2, a protein involved in nucleotide excision repair (NER), associates with the E3 ubiquitin ligase complex Cul4A-DDB1. But the precise role of these interactions in the NER activity of DDB2 is unclear. Several models, including DDB2-mediated ubiquitination of histones in UV-irradiated cells, have been proposed. But those models lack clear genetic evidence. Here we show that DDB2 participates in NER by regulating the cellular levels of p21(Waf1/Cip1). We show that DDB2 enhances nuclear accumulation of DDB1, which binds to a modified form of p53 containing phosphorylation at Ser18 (p53(S18P)) and targets it for degradation in low-dose-UV-irradiated cells. DDB2(-/-) mouse embryonic fibroblasts (MEFs), unlike wild-type MEFs, are deficient in the proteolysis of p53(S18P). Accumulation of p53(S18P) in DDB2(-/-) MEFs causes higher expression p21(Waf1/Cip1). We show that the increased expression of p21(Waf1/Cip1) is the cause NER deficiency in DDB2(-/-) cells because deletion or knockdown of p21(Waf1/Cip1) reverses their NER-deficient phenotype. p21(Waf1/Cip1) was shown to bind PCNA, which is required for both DNA replication and NER. Moreover, an increased level of p21(Waf1/Cip1) was shown to inhibit NER both in vitro and in vivo. Our results provide genetic evidence linking the regulation of p21(Waf1/Cip1) to the NER activity of DDB2.

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.

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 G1 phase is associated with a cyclin-dependent binding of the cyclin-dependent 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 in extracts of a cell line that expresses a temperature-sensitive mutant of p53. Incubation at the permissive temperature (32 degrees 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 speculated that the p21-mediated disruption of the cdk2-containing E2F-p130 complex plays a role in the growth suppression function of p53.

The transcription factor E2F has been shown to be involved in the expression of several cell cycle-regulated genes, and the activity of this factor is controlled by cellular proteins such as pRB and p107. E2F is also a target of the DNA virus oncoproteins (adenovirus E1A, simian virus 40 T antigen, and human papillomavirus [HPV] E7) (see the review by J. R. Nevins [Science 258: 424-429, 1992]). These viral oncoproteins dissociate an inactive complex between E2F and the retinoblastoma tumor suppressor protein (pRB), and this dissociation of the E2F-pRB complex correlates with a stimulation of the E2F-dependent transcription. In the S phase of the cell cycle, E2F forms a complex with p107, cyclin A, and the cdk2 kinase (E2F-cyclin A complex). The cellular function of this S-phase-specific complex is unclear. The adenovirus E1A protein dissociates the E2F-cyclin A complex. The HPV type 16 (HPV-16) E7 protein, which possesses significant sequence homology with E1A, does not dissociate the E2F-cyclin A complex. We find that the HPV-16 E7 protein associates very efficiently with the E2F-cyclin A complex. This association is dependent on the sequences that are also necessary for the transforming activity of E7. Moreover, the E7 protein of a low-risk HPV (type 6b) is much less efficient in binding to the E2F-cyclin A complex compared with that of the high-risk type. We also find that the E2F-cyclin A complex remains endogenously associated with the E7 protein in extracts of Caski cells, which express high levels of HPV-16 E7 protein. Finally, we have extensively purified the E2F-cyclin A complex from mouse L-cell extracts and show that, in cell extracts, the E2F-cyclin A complex remains associated with other cellular proteins.

Several reports have indicated that the product of the retinoblastoma gene (Rb) complexes with the transcription factor E2F. We present evidence that the DNA-binding of the Rb-E2F complex involves another cellular factor. Addition of Rb to purified preparations of E2F does not generate an Rb-E2F complex that can bind DNA, and in fact, we see an inhibition of the DNA-binding ability of E2F. On the other hand, addition of Rb to cruder preparations of E2F results in the formation of an Rb-E2F complex (E2Fr) that can bind DNA and produces a distinct complex in gel retardation assays. We have identified and purified a 60-kDa protein that allows the Rb-E2F complex to bind DNA, and we show that this 60-kDa protein exerts its effect by directly interacting with Rb.

Previous experiments have identified an element in the adenovirus E4 promoter that is critical for E1A-dependent trans activation and that can confer inducibility to a heterologous promoter. This DNA element is a recognition site for multiple nuclear factors, including ATF, which is likely a family of DNA-binding factors with similar DNA recognition properties. However, ATF activity was found not to be altered in any demonstrable way as a result of adenovirus infection. In contrast, another factor that recognizes this element, termed E4F, was found at only very low levels in uninfected cells but was increased markedly upon adenovirus infection, as measured in DNA-binding assays. Although both the ATF activity and the E4F activity recognized and bound to the same two sites in the E4 promoter, they differed in their sequence recognition of these sites. Furthermore, E4F bound only to a small subset of the ATF recognition sites; for instance, E4F did not recognize the ATF sites in the E2 or E3 promoters. Various E4F and ATF binding sites were inserted into an expression vector and tested by cotransfection assays for responsiveness to E1A. We found that a sequence capable of binding E4F could confer E1A inducibility. In contrast, a sequence that could bind ATF but not E4F did not confer E1A inducibility. We also found that E4F formed a stable complex with the E4 promoter, whereas the ATF DNA complex was unstable and rapidly dissociated. We conclude that the DNA-binding specificity of E4F as well as the alterations in DNA-binding activity of E4F closely correlates with E1A stimulation of the E4 promoter.

The promoter-specific factor E2F interacts with critical regulatory sequences within the adenovirus E2 promoter. In addition, the level of active factor increases markedly during a virus infection, dependent on E1A function and coincident with the trans activation of E2 transcription. We have purified the E2F factor through a combination of standard biochemical procedures and DNA affinity chromatography. The purified factor was a single polypeptide of 54,000 molecular weight, as determined by UV crosslinking and renaturation of gel-fractionated protein. Addition of affinity-purified factor to an in vitro transcription system resulted in stimulation of transcription from a promoter containing two E2F-binding sites but not promoters lacking binding sites. We thus conclude that E2F is indeed capable of stimulating transcription once it has bound to the promoter.