The Naturally Occurring Mutants of DDB Are Impaired in Stimulating Nuclear Import of the p125 Subunit and E2F1-Activated Transcription

Abstract

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...

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

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UVDDB p127-binding modulates activities and intracellular distribution of Hepatitis B virus X protein

“…In this respect, our ®nding that class 1 gain-of-binding mutant accumulated in the cell nucleus, in contrast to loss-of-binding class 3 mutants which remained cytoplasmic, supports the existence of a nuclear translocation step of the viral protein that would depend on its association with UVDDB. Interestingly, UVDDB itself, that alike X lacks a nuclear translocation signal, was shown to be translocated to the nucleus only when bound to another protein (DDB2p48) (Shiyanov et al, 1999). Taken together with our own data, these observations may provide an explanation for the rather con¯icting results reported for the subcellular distribution of wild type X protein (Dandri et al, 1998;Doria et al, 1995;Su et al, 1998).…”

Potential attenuation of p38 signaling by DDB2 as a factor in acquired TNF resistance

“…Since DDB2 is a transcription partner of E2F1, a cell cycle regulator. 24 DDB2 may participate in the transactivation of a target gene, whose product in turn regulates the p38 pathway. Preliminary results showed that DDB2 can activate the expression of an anti-apoptotic gene, cFLIP (our unpublished data), which is involved in the regulation of p38 signaling 25 and of caspase-8-dependent apoptosis.…”

“…4b, lanes 1-12). Inversely concomitant changes of the nuclear NF-kB, namely, a significant increase and subsequent decrease, occurred in each treated cells (lanes [17][18][19][20][21][22][23][24]. Taken together, DDB2 overexpression was insufficient to up-regulate NF-kB signal, and nor could it enhance the TNF-a-induced NF-kB signal.…”