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J. Biol. Chem., Vol. 275, Issue 28, 21429-21434, July 14, 2000

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Nuclear Transport of Human DDB Protein Induced by Ultraviolet Light^*

Wei Liu, Anne F. Nichols, Jay A. Graham, Rachel Dualan, Alex Abbas, and Stuart Linn

From the Division of Biochemistry and Molecular Biology, University of California, Berkeley, California 94720-3202

Received for publication, February 4, 2000, and in revised form, April 17, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Human damage-specific DNA-binding (DDB) protein can be purified as a heterodimer (p48 and p127) that binds to DNA damaged by ultraviolet light. We report here the effects of UV irradiation on the cellular localization of each DDB subunit as a function of time using green fluorescent fusion proteins in three diploid fibroblast strains: repair-proficient IMR-90 and two repair-deficient xeroderma pigmentosum group E strains (XP95TO and XP3RO). Although p48 remained in the nucleus after UV irradiation, a dynamic nuclear accumulation of p127 from the cytoplasm was found after 24 h. In IMR-90 cells, the nuclear localization of p127 corresponded to the up-regulation of p48 mRNA and protein levels and of DDB activity. XP3RO cells showed delayed but similar kinetics with less transport, whereas XP95TO cells appeared to have different kinetics, suggesting that these cells exhibit different defects in p127 translocation. We propose that p48 might act as the transporter for nuclear entry of p127 but that a third factor might be necessary for efficient transportation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Human damage-specific DNA-binding protein (DDB)¹ was originally identified as a nuclear factor that recognizes ultraviolet light (UV)-damaged DNA (1, 2). DDB activity is absent in cell strains from a subset (Ddb) of xeroderma pigmentosum group E (XPE) patients (2-4). DDB purifies from HeLa cells as a heterodimer of 127-kDa (DDB1) and 48-kDa (DDB2) subunits (5) and when microinjected into XPE cells, restores in vivo DNA repair synthesis to normal levels in XPE Ddb strains but not in XPE Ddb⁺ strains or those from other XP groups (6). In addition to its high affinity for UV-damaged DNA, DDB also recognizes a wide spectrum of DNA lesions (1, 2, 7-10), and the level of its activity is enhanced by treatment of cells with DNA-damaging agents (8, 10, 11). This increase appears to be due to induction of p48 but not p127 (12). It has been proposed that DDB has a DNA repair function, possibly acting in damage recognition prior to nucleotide excision repair (NER). However, NER reconstitution studies in vitro using purified protein components exhibit no requirement for DDB (13-15), and the induction of DDB by UV irradiation occurs subsequent to DNA repair (12). Therefore the exact in vivo biological function(s) of DDB still remain unknown.

cDNA sequence analysis indicates that the lack of DDB activity in XPE Ddb cells is due to mutations in DDB2 (p48) in strains XP2RO, XP3RO, XP82TO, and GM03189 (12, 16). No mutations in DDB1 (p127) have been found in XPE or any other cells. In an in vitro DDB activity assay, purified recombinant wild type p48 from insect cells was able to complement p127 to restore DDB activity to extracts from Ddb cells (12).

DDB p127 has been shown to be in excess of p48 in unirradiated HeLa cells (17). In normal diploid IMR-90 fibroblasts, the p48 mRNA copy number increases about 4-fold 38 h post-UV irradiation. A similar rise in p48 protein amount and DDB activity was also observed 48 h after irradiation (12). This change in DDB activity as a result of increased p48 protein is in agreement with the fact that there is excess p127 protein compared with p48 in uninduced cells.

Fluorescence microscopy using fusions of p48 and enhanced green fluorescent protein (GFP) indicated that p48 is localized in the nucleus in wild type, XPE Ddb⁺, and Ddb cells (12). DDB p127 has been found to redistribute into a tight association with chromatin shortly after UV irradiation, causing a decrease of extractable DDB activity (18). We have also observed the inhibition of DDB activity shortly after UV irradiation in our accompanying paper (12). In the present study, we have examined the effect of UV irradiation on the localizations of DDB subunits in both wild type and XPE cells as a function of time. The results of these experiments imply that p48 might act as the mediator of nuclear accumulation of p127.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cultures and Strains-- Human normal diploid lung fibroblast strain IMR-90 and two XPE fibroblast strains, XP3RO (Ddb) and XP95TO (Ddb⁺), were cultured as described previously (4, 16).

Biotinylated Lectin Assay-- Partially purified HeLa DDB (40-100 units) was resolved by electrophoresis on an 8% SDS-polyacrylamide gel and transferred onto a Protran BA85 nitrocellulose membrane (Schleicher and Schuell). The filter was probed with a biotinylated lectin kit (Vector) according to the manufacturer's directions. Lectins used were peanut agglutinin (PNA), Dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA), Ulex europaeus agglutinin (UEA), concanavalin A (ConA), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA). The membranes were incubated with 5 µg of biotinylated lectin per ml of TBST buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) with gentle rocking at room temperature for 30 min. After three 15-min washes with TBST, the membranes were incubated for 30 min with 0.2 µl of avidin-horseradish peroxidase conjugate (diluted 1:5000) per ml of TBST buffer, followed by three 15-min TBST washes. The membranes were soaked for 1 min in NEN Renaissance chemiluminescent solution and then exposed to x-ray film (Kodak) for 15-60 s, after which the autoradiographs were scanned with a Umax Astra 2400S scanner.

GFP Fluorescence Microscopy-- Mammalian expression vector pEGFP-N3, coding the mammalian codon-optimized, enhanced GFP, was obtained from CLONTECH. Full-length p127 cDNA was amplified by polymerase chain reaction as two segments using native pfu DNA polymerase (Stratagene) with two sets of primers: 5'-GCTGGATCCGCCACCATGTCGTACAACTACGTG-3' (forward) and 5'-GCTGATCTGCCGGAGCTCCTG-3' (reverse) for cDNA nucleotides +1 to +1590, and 5'-ATCCATCCTCAGGAGCTCCGG-3' (forward) and 5'-TGCCCTGGATCCATGGATTATAGTTAGCTC-3' (reverse) for cDNA nucleotides +1560 to +3420, respectively. The two cDNA segments were digested with BamHI and SacI and cloned into pEGFP-N3 at the BamHI site by a three-way ligation at 4 °C overnight to form pEGFP-p127. The GFP protein was consequently expressed as a fusion to the C terminus of p127. Full-length p48 cDNA was amplified in the same way as that of p127 with a primer set 5'-ACAGGATCCGCCACCATGGCTCCCAAGAAACGC-3' (forward) and 5'-TGTGGATCCCTTCCGTGTCCTGGCTTCC-3' (reverse), corresponding to cDNA nucleotides 15 to +18 and +1263 to +1290, respectively, and cloned in at the BamHI site of pEGFP-N3 to form the pEGFP-p48 recombinant vector for the expression of the p48-pGFP fusion protein.

Cell growth, transfection, UV irradiation, and fluorescence microscopy of IMR-90 and XPE cells were performed essentially as described in the accompanying paper (12). Confocal laser scanning fluorescence microscopy was performed on a Zeiss 510 system. At each time point, 9 to 30 cells were randomly examined and photographed. Slides were coded, and the cellular localization was evaluated by seven independent scorers. Classifications included primarily cytoplasmic and weakly nuclear, equal distribution to both cytoplasmic and nuclear, primarily nuclear and weakly cytoplasmic, and strictly nuclear localization.

DDB Activity Assay-- The electrophoretic mobility shift DDB activity assay was performed essentially as described in the accompanying paper (12).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Nuclear Localization of DDB Subunits as Suggested by Lectin Probes-- In one approach to infer the cellular localization of the DDB subunits, HeLa DDB was probed with biotinylated lectins specific for various sugar modifications (Fig. 1 and Table I). A sample of partially purified DDB (Fig. 1C, lane 1) provided additional non-DDB protein bands in the sample to serve as positive controls for the lectins and to rule out potential nonspecific binding of the lectins. Because no signal was detected with the RCA and wheat germ agglutinin lectins with this fraction (Fig. 1B, lanes 1 and 3), a less pure fraction (Fig. 1C, lane 2) was also probed (Fig. 1B, lanes 2 and 4). Specific, non-DDB bands were detected in this fraction, demonstrating that the lectins were active but did not bind to either DDB p127 or p48. Interaction visualized with the other lectins (Fig. 1A) indicated that p48 contains - and -linked N-acetylgalactosamine, -linked fucose, and -linked mannose, whereas p127 contains -linked N-acetylgalactosamine, -linked fucose, and -linked mannose. The glycosylation patterns are consistent with a nuclear or nuclear matrix localization (19) and suggest that, when isolated as an active heterodimer, both DDB subunits have nuclear localizations. It also agrees with the initial discovery of DDB as a nuclear factor with specific affinity for UV-damaged DNA (2). Fluorescence spectroscopy studies using a p48-pGFP fusion protein, as well as pSORT program prediction (20) (version 6.4), have also demonstrated that p48 is localized in the nucleus (12). However, the pSORT program did not identify any potential nuclear localization signals on p127 and in fact predicted by a small marginal certainty that p127 would be localized in the cytoplasm.

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Analysis of the gylcosylations of DDB subunits with lectin probes. A, a side fraction of fraction VI (5) of the HeLa DDB heterodimer purification (containing 100 units of DDB activity) was resolved by 8% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, probed with biotinylated lectins, and developed as described under "Experimental Procedures." M, high molecular weight marker (Amersham Pharmacia Biotech) with molecular sizes indicated to the left in kilodaltons. B, lanes 1 and 3, 80 units of DDB activity of the fraction utilized in A; lanes 2 and 4, 40 units of DDB activity of the less pure fraction V (5) were probed as in A. A Benchmark protein ladder (Life Technologies, Inc.) provided the positions indicated to the right in kilodaltons. C, silver stain of the partially purified HeLa DDB proteins used. Lane 1, side fraction of fraction VI (5), 40 units of DDB activity; lane 2, fraction V (5), 30 units of DDB activity; M, a benchmark protein ladder (Life Technologies, Inc.) with molecular sizes indicated to the right in kilodaltons.

View this table: [in this window] [in a new window]   Table I Lectin affinity assay of DDB subunits Purified HeLa DDB was probed with biotinylated lectins as described in Fig. 1, and the immunoreactivity of p48 and p127 bands were scored as positive (+) or negative (). Sugar specificity and binding site preference data were from Hart et al. (20).

DDB p127 Is Localized to Both the Nucleus and the Cytoplasm-- Because GFP protein alone does not show any preference for subcellular compartments, the fusion protein p127-pGFP can be used to study the localization of p127. Plasmids pEGFPN3 coding the GFP and pEGFP-p127 coding the p127-pGFP fusion protein were transfected into IMR-90 cells, and the localization of fluorescence signals was subsequently studied. In IMR-90 cells, GFP alone has a uniform whole cell distribution (Fig. 2A), whereas p127-pGFP has a primarily cytoplasmic, but weakly nuclear localization (Fig. 2B). Although previous studies reported that p127 is strictly localized to the cytoplasm of human osteoblastoma U2OS cells that have a low level of DDB (21), these data indicate that p127 is present in both the nucleus and cytoplasm in normal diploid fibroblasts. The GFP signal from the nucleus is not due to an overlaying signal from the cytoplasm because confocal laser scanning fluorescence microscopy essentially observed the same p127 localization pattern across different planes of the viewing field (Fig. 2C). The primarily cytoplasmic localization of p127 has also been demonstrated by the immunostaining of cytoplasmic and nuclear fractions of mouse PC-12 cells, as well as by immunofluorescence of p127 in human embryonic kidney 293 cells (22). Occasionally we observed p127-pGFP signals as a uniform whole cell distribution in a small fraction of cells (Fig. 3A). This observation could be due to the weaker expression of p127-pGFP in these particular cells.

View larger version (70K): [in this window] [in a new window]   Fig. 2.   Fluorescence microscopy analysis of DDB p127 localization in IMR-90 cells. IMR-90 cells were transfected with appropriate plasmids, UV-irradiated, and fixed as described under "Experimental Procedures." Cells were examined with a fluorescence microscope at 40× magnification and appropriate filters for GFP and 4,6-diamidino-2-phenylindole (DAPI). A, control (unfused GFP), no UV irradiation; B, p127-pGFP fusion, no UV irradiation; C, a confocal image of p127-pGFP, no UV irradiation; D, an image of IMR-90 cells showing increased nuclear accumulation of p127-pGFP 72 h after UV irradiation; E, an image of IMR-90 cells showing only nuclear localization of p127-pGFP 48 h after UV irradiation. Cells that give uniform distribution of the p127-pGFP fusion have the same signal pattern as that of control GFP alone (A) and are not shown here.

View larger version (34K): [in this window] [in a new window]   Fig. 3.   Nuclear accumulation of DDB p127 after UV irradiation. Experimental conditions are the same as those of Fig. 2. Localization of p127 is categorized as primarily cytoplasmic and weakly nuclear (open bar), equal distribution to both cytoplasmic and nuclear (hatched bar), primarily nuclear and weakly cytoplasmic (striped bar), and strictly nuclear localization (solid bar). At each time point, 9 to 30 cells were randomly examined and photographed. The pattern in each category is represented as a percentage of total cells examined. A, IMR-90 cells; B, XP95TO (Ddb+) cells; C, XP3RO (Ddb) cells.

Nuclear Translocation of p127 in IMR-90 Cells Induced by UV Irradiation-- To examine the effects of UV irradiation on the localization of each DDB subunit in normal diploid IMR-90 cells, the cells were transiently transfected with either control vector pEGFPN3 or the pEGFP-p48 or pEGFP-p127 recombinant constructs and then irradiated with 254 nm UV light at 10 J/m². The localization of fluorescence was then examined 24, 48, and 72 h post-UV irradiation (Fig. 3A). As expected, GFP protein alone had a uniform whole cell distribution, and UV irradiation had no effect on this pattern. Moreover, the p48 nuclear localization was unchanged by UV irradiation at all time points examined (data not shown).

On the other hand, a dynamic nuclear transportation of p127 was observed after UV irradiation. Before irradiation, p127-pGFP fluorescence was primarily cytoplasmic, with some cells (22% of all cells examined) showing a uniform distribution. However, 24 h after irradiation, the p127 localization pattern was reversed: 41% of the cells examined displayed primarily nuclear but weak cytoplasmic localization (Fig. 2D) whereas 14% of the cells showed strictly nuclear localization (Fig. 2E), and the rest (45%) exhibited a uniform distribution of p127-pGFP. Forty-eight h after UV irradiation, 70% of the cells had either primarily or strictly nuclear localization of p127-pGFP, with only 30% of the cells showing uniform distribution, and no primarily cytoplasmic localization of p127-pGFP was found. Seventy-two h after irradiation, all of the cells examined exhibited either primarily or strictly nuclear localization of p127-pGFP. The result of p127 subcellular translocation upon UV irradiation of normal IMR-90 fibroblasts is summarized in Fig. 3A. Interestingly, the occurrence of the translocation process correlated with the time course of increased p48 mRNA copy number, p48 protein amount, and DDB activity after UV irradiation described in the accompanying paper (12).

Effects of UV Irradiation on DDB Localization in XPE Cells-- The cellular localization of each DDB subunit in Ddb⁺ XP95TO and Ddb XP3RO strains was also examined. Cells were transiently transfected with either control vector pEGFPN3 or the pEGFP-p48 or pEGFP-p127 recombinant constructs and then irradiated with 254 nm UV light at 10 J/m². In both strains, GFP protein alone had a uniform distribution and p48 had a strictly nuclear localization, and as in normal IMR-90 cells, UV irradiation did not change these localization patterns (data not shown). We also observed that p127 had a primarily cytoplasmic and weak nuclear localization in the XPE cells, and as we had observed in IMR-90 cells, there were also a small number of cells (Fig. 3, B and C) that showed a uniform distribution of p127-pGFP in both strains, which again could be due to weaker expression of the fusion protein in these particular cells.

A nuclear accumulation of DDB p127 after UV irradiation was also observed in the XPE cells, but XP95TO and XP3RO had different kinetics of p127 nuclear transportation, and the transportation process was compromised in both strains as compared with that in IMR-90 cells (Fig. 3). For the XP95TO Ddb⁺ strain, cells exhibiting either primarily or strictly nuclear localization of p127 increased steadily from 11% of all cells examined after 24 h to 30% after 48 h to 75% after 72 h. But for the XP3RO Ddb strain, it increased from 30% after 24 h, peaked at 50% after 48 h, and dropped to 36% after 72 h. These results suggest that there could be different defects in the nuclear transportation of DDB p127 in these two XPE strains.

Because XP95TO cells have normal DDB activity, we also examined the induction of DDB activity after UV irradiation by the electrophoretic mobility shift assay (EMSA) (12). Compared with IMR-90 cells, the XP95TO cells showed a similar induction pattern of DDB activity after irradiation but possibly to a slightly lower extent (Fig. 4).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Induction of DDB activity by UV irradiation. IMR-90 and XP95TO cells were grown, UV-irradiated, and harvested, and cells extracts were prepared for EMSA assay as described in the accompanying paper (12). , IMR-90 cells; , XP95TO cells.

Possible p127 Anchoring Proteins in the Cytoplasm-- Watanabe et al. (22) demonstrated that DDB p127 can bind tightly to the cytoplasmic domain of the Alzheimer's amyloid precursor protein (APP_C) as judged by affinity co-purification studies: glutathione S-transferase-50 amino acid APP_C fusion peptide bound to DDB p127 from the cytoplasmic fraction of rat PC-12 cells. Such binding not only confirmed the cytosolic localization of DDB p127 but also suggested that APP_C might act to anchor p127 at the cytosolic side of the membrane. However, our confocal laser scanning fluorescence studies did not reveal a typical "ring" or "rim" pattern signal for membrane localization of p127-pGFP (Fig. 2C). Therefore, the p127-APP_C interaction would not appear to reflect an anchoring function, although it might have other yet unidentified functions for p127 or APP_C action. However, it is feasible that most of the APP_C is complexed with endogenous nonfluorescent p127 and that there is not additional APP_C available to bind a majority of the overexpressed p127-pGFP. Consequently, a rim pattern would not be observed.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

UV-induced Nuclear Localization of DDB p127-- DDB has a strong affinity for binding various DNA damages (1, 2, 5-10), and shortly after UV treatment, p127 has been reported to redistribute into a tight association with chromatin in mammalian cells (18). Such tight association with chromatin, which also occurs for nucleotide excision repair (NER) proteins such as RP-A and proliferating cell nuclear antigen, has been attributed to the binding specificity of DDB for damaged DNA and its possible role in damaged DNA recognition during initial steps of NER. In the present work we have demonstrated that the nuclear enrichment of DDB p127 occurs at times subsequent to DNA repair whereas p48 was observed to have a strict nuclear localization that was not affected by UV irradiation. These observations suggest possible roles for DDB protein and/or its individual subunits in addition to recognizing damaged DNA.

DDB p48 Mediates p127 Nuclear Localization-- Human DDB can be purified from HeLa cells as a heterodimer of p48 and p127 subunits (5). However, there is a molar excess of p127 over p48 in unirradiated HeLa cells (17). Previous experiments also indicated that the constitutive DDB activity in primate cells was induced to higher levels in UV-irradiated primate cells (8). The increase in DDB activity was then demonstrated to be due to an elevation in the amount of p48 protein because both subunits are needed for the binding to damaged DNA (12). In log-phase normal diploid fibroblast IMR-90 cells, UV irradiation at 12 J/m² caused a roughly 4-fold increase of DDB p48 mRNA level at 38 h after UV irradiation, followed by a similar rise in p48 protein amount and DDB activity after 48 h (12). This time course correlates with the dynamic nuclear translocation of p127 upon UV irradiation; while the majority of p127 is located in the cytoplasm in undamaged cells, the nuclear accumulation of p127 peaks between 24 and 48 h after UV irradiation. Such a correlation suggests that DDB p48 may also function as the transporter for p127 nuclear localization.

This proposal agrees with observations of Shiyanov et al. (21) that in human osteoblastoma U2OS cells that have very low constitutive levels of DDB transient overexpression of p48 caused increased nuclear accumulation of p127 (21). It also provides an explanation of their observation that DDB p127 was localized only to the cytoplasm in U2OS cells, because the low DDB level in those cells could be due to a small amount or a lack of DDB p48 protein or the presence of a mutant form of p48 that is inefficient or incapable of transporting p127. In normal diploid IMR-90 cells, however, the constitutively expressed level of p48 would be capable of escorting a significant fraction of the available p127 to the nucleus.

Possible Different Defects of p127 Nuclear Translocation in XPE Ddb+ and Ddb^- Cells-- In unirradiated XP95TO and XP3RO cells, DDB p127 also presents a primarily cytosolic but weakly nuclear localization pattern as seen in IMR-90 cells. In XP95TO Ddb⁺ cells, this pattern is consistent with the presence of the wild type p48 (12) that is capable of transporting p127 into the nucleus. In XP3RO Ddb cells, we expect that the roughly normal level of nuclear localization of p127 before irradiation could be due to the weak interaction of mutant p48 with p127, which was not stable enough for in vitro DDB activity assays (12) or in vitro immunoaffinity purifications (21) but is sufficient for escorting the nuclear entry of p127. This weaker interaction might also account for the fewer number of cells having a uniform distribution of p127 in XP3RO (13% of total cells examined) compared with those of XP95TO (17%) or IMR-90 (23%) cells.

While the in vitro activity assay showed that DDB activity was also induced in irradiated XP95TO cells as in IMR-90 cells (Fig. 4), the nuclear accumulation of p127 in XP95TO cells had different kinetics from that in IMR-90 cells: instead of reaching a plateau between 24 and 48 h after irradiation, the fraction of cells that showed either primarily or strictly nuclear localization of p127 increased steadily over time. These observations could suggest that, although p48 is normal in these cells, a possible third factor that is required for the efficient nuclear entry of p127 is defective. XP3RO cells, on the other hand, had similar but delayed kinetics of p127 nuclear accumulation compared with IMR-90 cells: the fraction of cells that displayed either primarily or strictly nuclear localization of p127 plateaued at 48 h after irradiation, although to a lower extent (50%) as compared with IMR-90 cells (70%). This delayed and inefficient nuclear entry of p127 in XP3RO could again be attributed to the mutation in the p48 subunit. Therefore, these data suggest that there may be different mutations that account for the inefficient nuclear accumulation of p127 after UV irradiation in Ddb⁺ versus Ddb XPE strains.

Effects of Protein-Protein Interactions on DDB Localization and Function-- DDB p48 has been shown to interact with the transcription factor E2F1, and such interaction can overcome the retinoblastoma protein (Rb)-mediated inhibition of E2F1-activated transcription (23). The association of p48 with CUL-4A (17) suggests an additional role in cell cycle regulation by DDB because the cullin family of proteins is believed to regulate the cell cycle by destroying cell cycle regulators via ubiquitination (24, 25). The up-regulation of only p48 expression after UV irradiation correlates with the fact that there is a molar excess of p127 compared with p48 in the cell. This excess of p127 found mainly in the cytoplasm as we have shown herein might have other yet unidentified distinct cytoplasmic functions. Such functions could, for example, possibly act in conjunction with the amyloid precursor protein (APP_C), although we have been unable to confirm a docking function of APP_C for DDB p127.

We have used a yeast two-hybrid system to identify other candidates for human proteins that interact with DDB p127. Preliminary studies isolated positive clones that encode segments of four different proteins: the BRCA1-associated protein 2, BRAP2 (26); a human homolog of the 26-kDa Xenopus RP-A interacting protein, XRIP (27); EBNA2 protein of the human Epstein-Barr virus (28); and subunit 3 of the COP9 signal transduction complex, Sgn3 (29).²

The C-terminal portion of BRAP2, which interacts with DDB p127 in the screen, has typical leucine heptad repeats for protein-protein interaction. BRAP2 is mainly cytoplasmic and binds to nuclear localization signal motifs in BRCA1, SV40 large T antigen, and mitosin. Because BRAP2 does not cycle between the nucleus and the cytoplasm, it has been proposed to be a cytoplasmic retention protein that plays a role in regulating nuclear transport of its interacting proteins (26). Therefore, DDB p127 could possibly be retained in the cytoplasm by BRAP2 until the up-regulation of p48.

The association of DDB with chromatin and RP-A shortly after UV irradiation (18) presumably reflects its role in recognizing damaged DNA. The human homolog of the Xenopus RP-A interacting protein that we identified in the screen might be involved in this association through its interaction with DDB p127 and RP-A. However, the delayed response to UV irradiation of up-regulating p48 expression and the nuclear accumulation of p127 indicate additional functions of newly transported DDB protein. Such functions could be the regulation of transcription and the cell cycle. In addition to the EBNA2 protein of the human Epstein-Barr virus, DDB p127 has been shown to interact with a variety of other viral transcription proteins, such as the X protein of the human hepatitis B virus (30), the V protein of the simian paramyxovirus SV5, the mumps virus, the human parainfluenza virus 2, and the measles virus (31). Furthermore, p127 has been reported to be structurally and immunologically highly related to the human lipoprotein B gene regulatory factor-2 (BRF-2) (32).

The possible interaction of DDB p127 with subunit 3 of the COP9 signal transduction complex, which is involved in cell cycle regulation, again suggests an additional role for DDB. Therefore, DDB may, in conjunction with other proteins, regulate cellular transcription process and control cell cycle progression after sensing extensive DNA damage. Experiments to verify and expand upon the preliminary results of the yeast two-hybrid screen are in progress.

	ACKNOWLEDGEMENTS

We thank Jill Fuss for help with confocal fluorescence microscopy and Ann Fischer for her expert culturing of cells. Fluorescence microscopy was performed at the CNR Center for Biological Imaging of the University of California at Berkeley.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grant P30ES08196 and Department of Energy Contract FG03-92ER61458.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Division of Biochemistry and Molecular Biology, 401 Barker Hall, University of California, Berkeley, CA 94720-3202. Tel.: 510-642-7583; Fax: 510-643-9290; E-mail: slinn@socrates.berkeley.edu.

Published, JBC Papers in Press, April 20, 2000, DOI 10.1074/jbc.M000961200

² R. Dualan, A. Abbas, and S. Linn, unpublished results.

	ABBREVIATIONS

The abbreviations used are: DDB, damage-specific DNA-binding protein; Ddb, absence of DDB activity; Ddb⁺, presence of DDB activity; XPE, xeroderma pigmentosum group E; PNA, peanut agglutinin; DBA, D. biflorus agglutinin; SBA, soybean agglutinin; UEA, U. europaeus agglutinin; ConA, concanavalin A; WGA, wheat germ agglutinin; RCA, R. communis agglutinin; NER, nucleotide excision repair; GFP, green fluorescent protein; EMSA, electrophoretic mobility shift assay; APP_C, the cytoplasmic domain of the Alzheimer's amyloid precursor protein; BRAP2, BRCA1-associated protein 2; EBNA2, Epstein-Barr virus nuclear antigen protein 2.

	REFERENCES

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				W. M. Al Khateeb and D. F. Schroeder DDB2, DDB1A and DET1 Exhibit Complex Interactions During Arabidopsis Development Genetics, May 1, 2007; 176(1): 231 - 242. [Abstract] [Full Text] [PDF]

				B. Schrofelbauer, Y. Hakata, and N. R. Landau HIV-1 Vpr function is mediated by interaction with the damage-specific DNA-binding protein DDB1 PNAS, March 6, 2007; 104(10): 4130 - 4135. [Abstract] [Full Text] [PDF]

				K. Shimanouchi, K.-i. Takata, M. Yamaguchi, S. Murakami, G. Ishikawa, R. Takeuchi, Y. Kanai, T. Ruike, R. Nakamura, Y. Abe, et al. Drosophila Damaged DNA Binding Protein 1 Contributes to Genome Stability in Somatic Cells J. Biochem., January 1, 2006; 139(1): 51 - 58. [Abstract] [Full Text] [PDF]

				M. A. Bevilacqua, B. Iovine, N. Zambrano, C. D'Ambrosio, A. Scaloni, T. Russo, and F. Cimino Fibromodulin Gene Transcription Is Induced by Ultraviolet Irradiation, and Its Regulation Is Impaired in Senescent Human Fibroblasts J. Biol. Chem., September 9, 2005; 280(36): 31809 - 31817. [Abstract] [Full Text] [PDF]

				K.-i. Takata, H. Yoshida, M. Yamaguchi, and K. Sakaguchi Drosophila Damaged DNA-Binding Protein 1 Is an Essential Factor for Development Genetics, October 1, 2004; 168(2): 855 - 865. [Abstract] [Full Text] [PDF]

				C. Obuse, H. Yang, N. Nozaki, S. Goto, T. Okazaki, and K. Yoda Proteomics analysis of the centromere complex from HeLa interphase cells: UV-damaged DNA binding protein 1 (DDB-1) is a component of the CEN-complex, while BMI-1 is transiently co-localized with the centromeric region in interphase Genes Cells, February 1, 2004; 9(2): 105 - 120. [Abstract] [Full Text] [PDF]

				V. Rapic-Otrin, V. Navazza, T. Nardo, E. Botta, M. McLenigan, D. C. Bisi, A. S. Levine, and M. Stefanini True XP group E patients have a defective UV-damaged DNA binding protein complex and mutations in DDB2 which reveal the functional domains of its p48 product Hum. Mol. Genet., July 1, 2003; 12(13): 1507 - 1522. [Abstract] [Full Text] [PDF]

				M. E. Fitch, I. V. Cross, and J. M. Ford p53 responsive nucleotide excision repair gene products p48 and XPC, but not p53, localize to sites of UV-irradiation-induced DNA damage, in vivo Carcinogenesis, May 1, 2003; 24(5): 843 - 850. [Abstract] [Full Text] [PDF]

				A. F. Nichols, T. Itoh, F. Zolezzi, S. Hutsell, and S. Linn Basal transcriptional regulation of human damage-specific DNA-binding protein genes DDB1 and DDB2 by Sp1, E2F, N-myc and NF1 elements Nucleic Acids Res., January 15, 2003; 31(2): 562 - 569. [Abstract] [Full Text] [PDF]

				F. Zolezzi, J. Fuss, S. Uzawa, and S. Linn Characterization of a Schizosaccharomyces pombe Strain Deleted for a Sequence Homologue of the Human Damaged DNA Binding 1 (DDB1) Gene J. Biol. Chem., October 18, 2002; 277(43): 41183 - 41191. [Abstract] [Full Text] [PDF]

				J. Andrejeva, E. Poole, D. F. Young, S. Goodbourn, and R. E. Randall The p127 Subunit (DDB1) of the UV-DNA Damage Repair Binding Protein Is Essential for the Targeted Degradation of STAT1 by the V Protein of the Paramyxovirus Simian Virus 5 J. Virol., October 11, 2002; 76(22): 11379 - 11386. [Abstract] [Full Text] [PDF]

				F. Cong, J. Tang, B. J. Hwang, B. Q. Vuong, G. Chu, and S. P. Goff Interaction between UV-damaged DNA Binding Activity Proteins and the c-Abl Tyrosine Kinase J. Biol. Chem., September 13, 2002; 277(38): 34870 - 34878. [Abstract] [Full Text] [PDF]

				K.-i. Takata, G. Ishikawa, F. Hirose, and K. Sakaguchi Drosophila damage-specific DNA-binding protein 1 (D-DDB1) is controlled by the DRE/DREF system Nucleic Acids Res., September 1, 2002; 30(17): 3795 - 3808. [Abstract] [Full Text] [PDF]

				F. Bergametti, D. Sitterlin, and C. Transy Turnover of Hepatitis B Virus X Protein Is Regulated by Damaged DNA-Binding Complex J. Virol., June 5, 2002; 76(13): 6495 - 6501. [Abstract] [Full Text] [PDF]

				V. Rapic-Otrin, M. P. McLenigan, D. C. Bisi, M. Gonzalez, and A. S. Levine Sequential binding of UV DNA damage binding factor and degradation of the p48 subunit as early events after UV irradiation Nucleic Acids Res., June 1, 2002; 30(11): 2588 - 2598. [Abstract] [Full Text] [PDF]

				X. Chen, Y. Zhang, L. Douglas, and P. Zhou UV-damaged DNA-binding Proteins Are Targets of CUL-4A-mediated Ubiquitination and Degradation J. Biol. Chem., December 14, 2001; 276(51): 48175 - 48182. [Abstract] [Full Text] [PDF]

				A. Nag, A. Datta, K. Yoo, D. Bhattacharyya, A. Chakrabortty, X. Wang, B. L. Slagle, R. H. Costa, and P. Raychaudhuri DDB2 Induces Nuclear Accumulation of the Hepatitis B Virus X Protein Independently of Binding to DDB1 J. Virol., November 1, 2001; 75(21): 10383 - 10392. [Abstract] [Full Text] [PDF]

A. Nag, T. Bondar, S. Shiv, and P. Raychaudhuri The Xeroderma Pigmentosum Group E Gene Product DDB2 Is a Specific Target of Cullin 4A in Mammalian Cells Mol. Cell. Biol.,