DDB, a Putative DNA Repair Protein, Can Function as a Transcriptional Partner of E2F1

Abstract

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...

E2F regulates DDB2: consequences for DNA repair in Rb-deficient cells

“…It has previously been shown that DDB2 interacts with E2F1 and together with DDB1, stimulates E2F1-activated transcription (Hayes et al, 1998;Shiyanov et al, 1999, also reviewed by Tang and Chu, 2002). Our results support the hypothesis that in untransformed primary mouse epithelial cells, activation of DDB2 by E2F could be critical for the regulation of the G1/S transition.…”

Regulation of epidermal apoptosis and DNA repair by E2F1 in response to ultraviolet B radiation

“…In addition to transcriptionally regulating DNA repair genes, E2F1 also physically associates with a number of DNA repair proteins including DDB2, NBS1 and TopBP1 (Hayes et al, 1998;Maser et al, 2001;Liu et al, 2003). E2F1 has also been shown to colocalize with BRCA1 in foci that are presumptive sites of DNA repair .…”

“…These include transcriptional upregulation of DNA repair factors such as DDB2 and XPC (Adimoolam and Ford, 2002;Amundson et al, 2002;Tan and Chu, 2002;Fitch et al, 2003), recruitment of repair factors to sites of DNA damage through physical interaction with p53 (Wang et al, 2003) and the ability of p53 to function as a chromatin accessibility factor to allow access of repair factors to sites of damage (Rubbi and Milner, 2003). Given that E2F1 can upregulate the expression of a number of DNA repair genes (Wang et al, 1999;Polager et al, 2002;Ren et al, 2002) and that E2F1 physically associates with several DNA repair factors (Hayes et al, 1998;Maser et al, 2001;Liu et al, 2003), it is quite possible that like p53, the antiapoptotic effect of E2F1 is related to a stimulation of DNA repair.…”

Tumor-prone phenotype of the DDB2-deficient mice

“…In addition to its role in DNA repair, DDB2 has been also implicated in transcription. DDB2 associates with the transcriptional activator protein E2F1 (Hayes et al, 1998). Moreover, DDB2 also associates with the transcriptional coactivator proteins CBP/p300 and the STAGA complex (Datta et al, 2001;Martinez et al, 2001).…”