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• W2082157476 abstract "Human damaged DNA-binding protein (DDB) is a heterodimer of p48/DDB2 and p127/DDB1 subunits. Mutations in DDB2 are responsible for Xeroderma Pigmentosum group E, but no mutants of mammalian DDB1 have been described. To study DDB1, theSchizosaccharomyces pombe DDB1 sequence homologue (ddb1+) was cloned, and a ddb1deletion strain was constructed. The gene is not essential; however, mutant cells showed a 37% impairment in colony-forming ability, an elongated phenotype, and abnormal nuclei. The ddb1Δstrain was sensitive to UV irradiation, X-rays, methylmethane sulfonate, and thiabendazole, and these sensitivities were compared with those of the well characterized rad13Δ,rhp51Δ, and cds1Δ mutant strains. Ddb1p showed nuclear and nucleolar localization, and the aberrant nuclear structures observed in the ddb1Δ strain suggest a role for Ddb1p in chromosome segregation. Human damaged DNA-binding protein (DDB) is a heterodimer of p48/DDB2 and p127/DDB1 subunits. Mutations in DDB2 are responsible for Xeroderma Pigmentosum group E, but no mutants of mammalian DDB1 have been described. To study DDB1, theSchizosaccharomyces pombe DDB1 sequence homologue (ddb1+) was cloned, and a ddb1deletion strain was constructed. The gene is not essential; however, mutant cells showed a 37% impairment in colony-forming ability, an elongated phenotype, and abnormal nuclei. The ddb1Δstrain was sensitive to UV irradiation, X-rays, methylmethane sulfonate, and thiabendazole, and these sensitivities were compared with those of the well characterized rad13Δ,rhp51Δ, and cds1Δ mutant strains. Ddb1p showed nuclear and nucleolar localization, and the aberrant nuclear structures observed in the ddb1Δ strain suggest a role for Ddb1p in chromosome segregation. Human damaged DNA-binding protein (DDB) 1The abbreviations used are: DDB, Damaged DNA-binding protein, XP-E, Xeroderma Pigmentosum complementation group E; NER, nucleotide excision repair; ORF, open reading frame; DAPI, 4,6-diamono-2-phenylindole dihydrochloride; MMS, methylmethane sulfonate; HU, hydroxyurea; TBZ, thiabendazole; GFP, green fluorescent protein. 1The abbreviations used are: DDB, Damaged DNA-binding protein, XP-E, Xeroderma Pigmentosum complementation group E; NER, nucleotide excision repair; ORF, open reading frame; DAPI, 4,6-diamono-2-phenylindole dihydrochloride; MMS, methylmethane sulfonate; HU, hydroxyurea; TBZ, thiabendazole; GFP, green fluorescent protein. is a heterodimer of p48/DDB2 and p127/DDB1 subunits that binds to a variety of DNA lesions produced by UV light (1Dualan R. Brody T. Keeney S. Nichols A.F. Admon A. Linn S. Genomics. 1995; 29: 62-69Google Scholar, 2Keeney S. Chang G.J. Linn S. J. Biol. Chem. 1993; 268: 21293-212300Google Scholar, 3Reardon J.T. Nichols A.F. Keeney S. Smith C.A. Taylor J.S. Linn S. Sancar A. J. Biol. Chem. 1993; 268: 21301-21308Google Scholar, 4Fujiwara Y. Masutani C. Mizukoshi T. Kondo J. Hanaoka F. Iwai S. J. Biol. Chem. 1999; 274: 20027-20033Google Scholar). Mutations in DDB2 result in the elimination of DDB activity, giving rise to the disorder Xeroderma Pigmentosum complementation group E (XP-E) (5Chu G. Chang E. Science. 1988; 242: 564-567Google Scholar, 6Kataoka H. Fujiwara Y. Biochem. Biopyhs. Res. Commun. 1991; 175: 1139-1143Google Scholar, 7Keeney S. Wein H. Linn S. Mutat. Res. 1992; 273: 49-56Google Scholar, 8Nichols A.F. Itoh T. Graham J.A. Liu W. Yamaizumi M. Linn S. J. Biol. Chem. 2000; 275: 21422-21428Google Scholar, 9Itoh T. Mori T. Ohkubo H. Yamaizumi M. J. Invesy. Dermatol. 1999; 113: 251-257Google Scholar). No mutations have been found in mammalian DDB1.XP-E cells are only mildly defective in nucleotide excision repair (NER) of DNA damage, and microinjection of purified DDB corrects these deficiencies (10Keeney S. Eker A.P. Brody T. Vermeulen W. Bootsma D. Hoeijmakers J.H. Linn S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4053-4056Google Scholar). Moreover, DDB stimulates, but is not necessary for, NER in vitro (11Wakasugi M. Kawashima A. Morioka H. Linn S. Sancar A. Mori T. Nikaido O. Matsunaga T. J. Biol. Chem. 2002; 277: 1637-1640Google Scholar), but it appears to be necessary for normal global genomic NER (12Tang J.Y. Hwang B.J. Ford J.M. Hanawalt P.C. Chu G. Mol. Cell. 2000; 5: 737-744Google Scholar). It associates with the histone acetyltransferase, cAMP-response element-binding protein-binding protein/p300, which is believed to be important in altering chromatin structure. Datta et al. (13Datta A. Bagchi S. Nag A. Shiyanov P. Adami G.R. Yoon T. Raychaudhuri P. Mutat. Res. 2001; 486: 89-97Google Scholar) proposed that DDB has a stimulatory role in normal global genomic NER of UV-induced DNA lesions by recruiting cAMP-response element-binding protein-binding protein/p300 to the site of damage to render chromatin more accessible to the DNA repair machinery.DDB interacts with other cellular proteins, suggesting that it may be multifunctional. DDB2 binds to the cell cycle regulatory transcription factor, E2F1, and in the presence of DDB1 it acts as a negative regulator of G1/S cell cycle progression following UV-induced DNA damage (14Hayes S. Shiyanov P. Chen X. Raychaudhuri P. Mol. Cell. Biol. 1998; 18: 240-249Google Scholar). Both DDB1 and DDB2 are ubiquitinated by Cul-4A, a member of the cullin family of proteins (15Shiyanov P. Nag A. Raychaudhuri P. J. Biol. Chem. 1999; 274: 35309-35312Google Scholar, 16Chen X. Zhang Y. Douglas L. Zhou P. J. Biol. Chem. 2001; 276: 48175-48182Google Scholar, 17Nag A. Bondar T. Shiv S. Raychaudhuri P. Mol. Cell. Biol. 2001; 21: 6738-6747Google Scholar). Cul-4A is believed to be a ubiquitin-protein isopeptide ligase (type E3) and is involved in G1/S phase progression inCaenorhabditis elegans (18Kipreos E.T. Lander L.E. Wing J.P., He, W.W. Hedgecock E.M. Cell. 1996; 85: 829-839Google Scholar). The expression of DDB2 is cell cycle-regulated, peaking at the G1/S boundary (17Nag A. Bondar T. Shiv S. Raychaudhuri P. Mol. Cell. Biol. 2001; 21: 6738-6747Google Scholar). DDB2, but not DDB1, mRNA and protein levels are induced 2- to 3-fold by UV irradiation (19Itoh T. Nichols A. Linn S. Oncogene. 2001; 20: 7041-7050Google Scholar).DDB1 binds to the apolipoprotein B (apoB) gene regulatory factor 2 (BRF-2), and the DDB1-BRF-2 heterodimer has been suggested to be required for optimum-specific apoB gene expression (20Krishnamoorthy R.R. Lee T.H. Butel J.S. Das H.K. Biochemistry. 1997; 36: 960-969Google Scholar). DDB1 also binds to viral transcriptional transactivators, including the hepatitis B virus X protein (HBVx) (21Butel J.S. Lee T.H. Slagle B.L. Princess Takamatsu Symp. 1995; 25: 185-198Google Scholar) and the V proteins from paramyxovirus, simian parainfluenza virus 5, mumps virus, human parainfluenza virus 2, and measles virus (22Lin G.Y. Lamb R.A. J. Virol. 2000; 74: 9152-9166Google Scholar). DDB2 also has been shown recently to bind HBVx protein (23Nag A. Datta A. Yoo K. Bhattacharyya D. Chakrabortty A. Wang X. Slagle B.L. Costa R.H. Raychaudhuri P. J. Virol. 2001; 75: 10383-10392Google Scholar). Because nuclear levels of DDB increase in late G1, DDB has been proposed to participate in nuclear functions of HBVx during the late G1 phase of the cell cycle. Despite this information, the precise physiological role(s) of DDB still remain elusive.We have identified three domains that are highly conserved in DDB1 sequence homologues of Homo sapiens, Mus musculus, Drosophila melanogaster, C. elegans, Dictyostelium discoideum,Arabidopsis thaliana, S. pombe (24Zolezzi F. Linn S. Gene. 2000; 245: 151-159Google Scholar), andOryza sativa. 2Unpublished data. 2Unpublished data. Two other reports assigned the same S. pombe predicted polypeptide to the DDB1 family of proteins (25Mintz P.J. Patterson S.D. Neuwald A.F. Spahr C.S. Spector D.L. EMBO J. 1999; 18: 4308-4320Google Scholar, 26Neuwald A.F. Poleksic A. Nucleic Acids Res. 2000; 28: 3570-3580Google Scholar). The overall identity between the human and S. pombe (previously published as NCBI accession number SPAC17H9.10c) sequences is 26%; the identities in domains 1, 2, and 3 are 45, 40, and 21%, respectively. To our knowledge, no sequence homologue of DDB1 has been found in Saccharomyces cerevisiae.S. pombe has been used extensively to study DNA repair and cell cycle control (27Humphrey T. Mutat. Res. 2000; 451: 211-226Google Scholar, 28McCready S.J. Osman F. Yasui A. Mutat. Res. 2000; 451: 197-210Google Scholar). S. pombe appears to be more closely related to animal cells with respect to cell cycle regulation and chromosome structure and segregation than is S. cerevisiae (29Zhao Y. Lieberman H.B. DNA Cell Biol. 1995; 14: 359-371Google Scholar) from which it diverged 330–420 million years ago (30Sipiczki M. Genome Biol. 2000; 1 (Reviews): 1011.1-1011.4Google Scholar). In this study we report the cloning of the S. pombesequence homologue of the human DDB1 gene,ddb1+, and the characterization of theddb1 deletion mutant. ddb1+ is not an essential gene, and the ddb1Δ cells show an elongated phenotype and nuclear abnormalities. The sensitivity of theddb1 deletion strain to a variety of DNA-damaging agents, to hydroxyurea, and to thiabendazole was compared with those of the null strains rad13 (the gene encoding for the homologue of the human XPG protein (31Edwards R.J. Carr A.M. Methods Enzymol. 1997; 283: 471-494Google Scholar)), rhp51 (the gene encoding for the homologue of the human RAD51 protein (32Muris D.F. Vreeken K. Carr A.M. Broughton B.C. Lehmann A.R. Lohman P.H. Pastink A. Nucleic Acids Res. 1993; 21: 4586-4591Google Scholar)), and cds1 (a gene implicated in the intra-S checkpoint response, the homologue of the human CHEK2 (33Boddy M.N. Furnari B. Mondesert O. Russell P. Science. 1998; 280: 909-912Google Scholar, 34Lindsay H.D. Griffiths D.J. Edwards R.J. Christensen P.U. Murray J.M. Osman F. Walworth N. Carr A.M. Genes Dev. 1998; 12: 382-395Google Scholar)).DISCUSSIONDDB from mammalian cells is a heterodimer of DDB1 and DDB2 (2Keeney S. Chang G.J. Linn S. J. Biol. Chem. 1993; 268: 21293-212300Google Scholar, 3Reardon J.T. Nichols A.F. Keeney S. Smith C.A. Taylor J.S. Linn S. Sancar A. J. Biol. Chem. 1993; 268: 21301-21308Google Scholar, 4Fujiwara Y. Masutani C. Mizukoshi T. Kondo J. Hanaoka F. Iwai S. J. Biol. Chem. 1999; 274: 20027-20033Google Scholar). As noted in the Introduction, DDB1 is an evolutionarily conserved protein; sequence homologues have been identified in mammals, worms, insects and plants. DDB2, however, is less conserved, and DDB2 sequence homologues have been identified only in mammals and plants (24Zolezzi F. Linn S. Gene. 2000; 245: 151-159Google Scholar). 3Unpublished data. Members of the DDB1 family do not contain nuclear localization signals, whereas all of the DDB2 homologues contain multiple nuclear localization signals (1Dualan R. Brody T. Keeney S. Nichols A.F. Admon A. Linn S. Genomics. 1995; 29: 62-69Google Scholar). 4Unpublished data. Nevertheless, when the Ddb1p-Myc fusion protein was expressed under the control of the natural chromosomal promoter, an almost exclusively nuclear/nucleolar localization was observed. In human cells, DDB2 is proposed to bind to DDB1 in the cytoplasm and translocate it to the nucleus (44Liu W. Nichols A.F. Graham J.A. Dualan R. Abbas A. Linn S. J. Biol. Chem. 2000; 275: 21429-21434Google Scholar, 54Shiyanov P. Hayes S.A. Donepudi M. Nichols A.F. Linn S. Slagle B.L. Raychaudhuri P. Mol. Cell. Biol. 1999; 19: 4935-4943Google Scholar). This hypothesis is supported by the finding that two naturally occurring XP-E mutations of DDB2, 82TO and 2RO, are deficient in stimulating the nuclear accumulation of DDB1 (54Shiyanov P. Hayes S.A. Donepudi M. Nichols A.F. Linn S. Slagle B.L. Raychaudhuri P. Mol. Cell. Biol. 1999; 19: 4935-4943Google Scholar). Therefore, one might expect there to be a functional homologue of human DDB2 in S. pombe that is responsible for the import of Ddb1p into the nucleus. Alternatively, it is also possible that a S. pombe DDB2 functional homologue does not exist. In mammalian cells, DDB2 is induced in response to UV irradiation (19Itoh T. Nichols A. Linn S. Oncogene. 2001; 20: 7041-7050Google Scholar), and DDB1 is subsequently transported to the nucleus. Moreover, this induction is dependent upon p53 (55Hwang B.J. Ford J.M. Hanawalt P.C. Chu G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 424-428Google Scholar). By contrast,S. pombe Ddb1p does not relocate following UV irradiation, because it is constitutively present in the nucleus, and S. pombe does not have a p53 homologue.The FZ150 ddb1Δ strain was slightly sensitive to UV irradiation compared with the NER-defective rad13Δ strain, suggesting that Ddb1p does not play a principal role in NER of UV-induced DNA damage. The FZ150 strain also showed a slight sensitivity to X-rays. In both cases, these cells showed the presence of one subpopulation of about 40% with a high sensitivity to the radiation and a second subpopulation with normal sensitivity. Thirty-nine percent of the FZ150 cell population also had an elongated phenotype, suggesting that these elongated cells could be the subpopulation that is sensitive to irradiation. However, because it appeared that roughly the same proportion of cells do not form colonies, this correlation may be coincidental as it may be that the elongated cells do not replicate.The basis of the sensitivity of the ddb1D cells to MMS remains unclear, because no increase in aberrant nuclei was detected following exposure to MMS in liquid culture. Possibly Ddb1p is involved in base excision repair, but then a sensitivity to H2O2 would also have been expected. It is interesting that the rhp51D strain is also sensitive to MMS but not to H2O2, suggesting a possible role of Ddb1p in recombinational repair rather than in excision repair.Ddb1p appears also to be required for proper chromosomal segregation under normal growth conditions. Observations of chromosome III with indirect immunofluorescence of the rDNA-associated protein, Nop1p, showed that the chromatin abnormalities observed in at least some of the ddb1Δ cells appeared to be because of chromatid non-disjunction or premature disjunction. Neuwald and Poleksic (26Neuwald A.F. Poleksic A. Nucleic Acids Res. 2000; 28: 3570-3580Google Scholar) used hidden Markov models of structural repeats to predict the presence of β-propeller domains in the DDB1 family of proteins and in other structurally related proteins, including the fission yeast Rik1p-silencing protein. rik1+ belongs to a class of S. pombe-silencing genes that includescrl4+, clr6+,swi6+, and hst4+. These genes have been implicated in silencing of the mating-type loci, the telomeres, the centromeres, and the rDNA repeats and also to be essential elements in the assembly of a heterochromatin-like structure (50Allshire R.C. Nimmo E.R. Ekwall K. Javerzat J.P. Cranston G. Genes Dev. 1995; 9: 218-233Google Scholar, 51Ekwall K. Javerzat J.P. Lorentz A. Schmidt H. Cranston G. Allshire R. Science. 1995; 269: 1429-1431Google Scholar, 52Ekwall K. Nimmo E.R. Javerzat J.P. Borgstrom B. Egel R. Cranston G. Allshire R. J. Cell Sci. 1996; 109: 2637-2648Google Scholar, 53Grewal S.I. Bonaduce M.J. Klar A.J. Genetics. 1998; 150: 563-576Google Scholar, 56Freeman-Cook L.L. Sherman J.M. Brachmann C.B. Allshire R.C. Boeke J.D. Pillus L. Mol. Biol. Cell. 1999; 10: 3171-3186Google Scholar). Mutations in rik1, as well as inclr4, clr6, swi6, and hst4, also result in a defective chromatin structure leading to abnormal chromosome segregation. Most similar to the ddb1Δphenotype is the hst4Δ phenotype. These cells show the same elongation and abnormal nuclear morphology as theddb1Δ cells (56Freeman-Cook L.L. Sherman J.M. Brachmann C.B. Allshire R.C. Boeke J.D. Pillus L. Mol. Biol. Cell. 1999; 10: 3171-3186Google Scholar). Moreover, like Ddb1p, Hst4p shows a preferential nucleolar localization, and the hst4Δ cells are sensitive to UV irradiation (56Freeman-Cook L.L. Sherman J.M. Brachmann C.B. Allshire R.C. Boeke J.D. Pillus L. Mol. Biol. Cell. 1999; 10: 3171-3186Google Scholar).Genomic instability in the rik1, clr4,clr6, swi6, and hst4 mutants is thought to be caused by a defect in centromeric silencing, resulting in defective centromeric function (50Allshire R.C. Nimmo E.R. Ekwall K. Javerzat J.P. Cranston G. Genes Dev. 1995; 9: 218-233Google Scholar, 51Ekwall K. Javerzat J.P. Lorentz A. Schmidt H. Cranston G. Allshire R. Science. 1995; 269: 1429-1431Google Scholar, 52Ekwall K. Nimmo E.R. Javerzat J.P. Borgstrom B. Egel R. Cranston G. Allshire R. J. Cell Sci. 1996; 109: 2637-2648Google Scholar, 53Grewal S.I. Bonaduce M.J. Klar A.J. Genetics. 1998; 150: 563-576Google Scholar). Because clr4,rik1, swi6, and hst4 deletion mutants are highly sensitive to the microtubule-destabilizing drug TBZ, a direct or indirect interaction of the products of these genes with microtubules at the kinetochore was proposed (52Ekwall K. Nimmo E.R. Javerzat J.P. Borgstrom B. Egel R. Cranston G. Allshire R. J. Cell Sci. 1996; 109: 2637-2648Google Scholar). Theddb1Δ strain is also sensitive to TBZ, indicating that theddb1Δ strain might also be defective in an interaction of the centromere with microtubules. Indeed the chromosomal segregation abnormalities observed in the ddb1Δ cells, the presence of lagging chromosomes, and the Ddb1p nucleolar localization, together with the structural homology proposed between Ddb1p and Rik1p, suggest that Ddb1p might help to maintain normal heterochromatin structure at centromeric regions and possibly also at rDNA repeats. Should this be the case, the absence of Ddb1p could result in mispositioning of other unidentified proteins and consequent improper centromere assembly and genomic instability. In this light, the sensitivity of theddb1Δ strain to a variety of DNA-damaging agents could be interpreted as the result of inefficient DNA repair because of a general chromatin assembly defect rather then a specific defect in a DNA repair pathway.The organization of rDNA genes and of the centromeric regions inS. pombe has been shown to be more closely related to that in animal cells than that in S. cerevisiae (47Maleszka R. Clark-Walker G.D. Yeast. 1993; 9: 53-58Google Scholar, 48Toda T. Nakaseko Y.,. Niwa O. Yanagida M. Curr. Genet. 1984; 8: 93-97Google Scholar, 57Allshire R.C. Cranston G. Gosden J.R. Maule J.C. Hastie N.D. Fantes P.A. Cell. 1987; 50: 391-403Google Scholar, 58Hoheisel J.D. Maier E. Mott R. McCarthy L. Grigoriev A.V. Schalkwyk L.C. Nizetic D. Francis F. Lehrach H. Cell. 1993; 73: 109-120Google Scholar, 59Mizukami T. Chang W.I. Garkavtsev I. Kaplan N. Lombardi D. Matsumoto T. Niwa O. Kounosu A. Yanagida M. Marr T.G. et al.Cell. 1993; 73: 121-132Google Scholar, 60Fan J.-B. Chikashige Y. Smith C.L. Niwa O. Yanagida M. Cantor C.R. Nucleic Acids Res. 1989; 17: 2801-2818Google Scholar, 61Barnitz J.T. Cramer J.H. Rownd R.H. Cooley L. Soll D. FEBS Lett. 1982; 143: 129-132Google Scholar, 62Clarke L. Trends Genet. 1990; 6: 150-154Google Scholar). A function for the S. pombe Ddb1p in maintaining proper chromatin structure in the rDNA genes and the centromeric regions might then explain why a DDB1 homologue is not present in S. cerevisiae but is found in S. pombe. Aravindet al. (63Aravind L. Watanabe H. Lipman D.J. Koonin E.V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11319-11324Google Scholar) compared 4,344 protein sequences from S. pombe with all available eukaryotic sequences and identified those genes that are conserved in S. pombe and non-fungal eukaryotes but are missing or highly diverged in S. cerevisiae. Their results suggested coelimination of functionally interacting sets of proteins. In particular, a pattern of coelimination was seen among proteins involved in chromatin remodeling, including Swi6p, Clr4p, Rik1p, Ddb1p, and the cullin 4A ortholog. As previously noted, both human DDB1 and DDB2 are ubiquitinated by Cullin 4A.A stimulus for this study was ultimately to obtain clues to the function of mammalian DDB1, because naturally occurring mutations of DDB1 are not known. Although it is not clearly established that the mammalian and S. pombe sequence homologues are functional homologues, as well, the very potent defects that result as a consequence of the S. pombe ddb1 deletion would suggest that DDB1 mutations in mammalian cells are likely to be lethal. Moreover, the chromosome segregation defect observed in theddb1Δ mutant might suggest that subtle alterations in the DDB1 gene such as those caused by single nucleotides polymorphisms could lead to genomic instability and therefore to increased susceptibility to tumorigenesis. Human damaged DNA-binding protein (DDB) 1The abbreviations used are: DDB, Damaged DNA-binding protein, XP-E, Xeroderma Pigmentosum complementation group E; NER, nucleotide excision repair; ORF, open reading frame; DAPI, 4,6-diamono-2-phenylindole dihydrochloride; MMS, methylmethane sulfonate; HU, hydroxyurea; TBZ, thiabendazole; GFP, green fluorescent protein. 1The abbreviations used are: DDB, Damaged DNA-binding protein, XP-E, Xeroderma Pigmentosum complementation group E; NER, nucleotide excision repair; ORF, open reading frame; DAPI, 4,6-diamono-2-phenylindole dihydrochloride; MMS, methylmethane sulfonate; HU, hydroxyurea; TBZ, thiabendazole; GFP, green fluorescent protein. is a heterodimer of p48/DDB2 and p127/DDB1 subunits that binds to a variety of DNA lesions produced by UV light (1Dualan R. Brody T. Keeney S. Nichols A.F. Admon A. Linn S. Genomics. 1995; 29: 62-69Google Scholar, 2Keeney S. Chang G.J. Linn S. J. Biol. Chem. 1993; 268: 21293-212300Google Scholar, 3Reardon J.T. Nichols A.F. Keeney S. Smith C.A. Taylor J.S. Linn S. Sancar A. J. Biol. Chem. 1993; 268: 21301-21308Google Scholar, 4Fujiwara Y. Masutani C. Mizukoshi T. Kondo J. Hanaoka F. Iwai S. J. Biol. Chem. 1999; 274: 20027-20033Google Scholar). Mutations in DDB2 result in the elimination of DDB activity, giving rise to the disorder Xeroderma Pigmentosum complementation group E (XP-E) (5Chu G. Chang E. Science. 1988; 242: 564-567Google Scholar, 6Kataoka H. Fujiwara Y. Biochem. Biopyhs. Res. Commun. 1991; 175: 1139-1143Google Scholar, 7Keeney S. Wein H. Linn S. Mutat. Res. 1992; 273: 49-56Google Scholar, 8Nichols A.F. Itoh T. Graham J.A. Liu W. Yamaizumi M. Linn S. J. Biol. Chem. 2000; 275: 21422-21428Google Scholar, 9Itoh T. Mori T. Ohkubo H. Yamaizumi M. J. Invesy. Dermatol. 1999; 113: 251-257Google Scholar). No mutations have been found in mammalian DDB1. XP-E cells are only mildly defective in nucleotide excision repair (NER) of DNA damage, and microinjection of purified DDB corrects these deficiencies (10Keeney S. Eker A.P. Brody T. Vermeulen W. Bootsma D. Hoeijmakers J.H. Linn S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4053-4056Google Scholar). Moreover, DDB stimulates, but is not necessary for, NER in vitro (11Wakasugi M. Kawashima A. Morioka H. Linn S. Sancar A. Mori T. Nikaido O. Matsunaga T. J. Biol. Chem. 2002; 277: 1637-1640Google Scholar), but it appears to be necessary for normal global genomic NER (12Tang J.Y. Hwang B.J. Ford J.M. Hanawalt P.C. Chu G. Mol. Cell. 2000; 5: 737-744Google Scholar). It associates with the histone acetyltransferase, cAMP-response element-binding protein-binding protein/p300, which is believed to be important in altering chromatin structure. Datta et al. (13Datta A. Bagchi S. Nag A. Shiyanov P. Adami G.R. Yoon T. Raychaudhuri P. Mutat. Res. 2001; 486: 89-97Google Scholar) proposed that DDB has a stimulatory role in normal global genomic NER of UV-induced DNA lesions by recruiting cAMP-response element-binding protein-binding protein/p300 to the site of damage to render chromatin more accessible to the DNA repair machinery. DDB interacts with other cellular proteins, suggesting that it may be multifunctional. DDB2 binds to the cell cycle regulatory transcription factor, E2F1, and in the presence of DDB1 it acts as a negative regulator of G1/S cell cycle progression following UV-induced DNA damage (14Hayes S. Shiyanov P. Chen X. Raychaudhuri P. Mol. Cell. Biol. 1998; 18: 240-249Google Scholar). Both DDB1 and DDB2 are ubiquitinated by Cul-4A, a member of the cullin family of proteins (15Shiyanov P. Nag A. Raychaudhuri P. J. Biol. Chem. 1999; 274: 35309-35312Google Scholar, 16Chen X. Zhang Y. Douglas L. Zhou P. J. Biol. Chem. 2001; 276: 48175-48182Google Scholar, 17Nag A. Bondar T. Shiv S. Raychaudhuri P. Mol. Cell. Biol. 2001; 21: 6738-6747Google Scholar). Cul-4A is believed to be a ubiquitin-protein isopeptide ligase (type E3) and is involved in G1/S phase progression inCaenorhabditis elegans (18Kipreos E.T. Lander L.E. Wing J.P., He, W.W. Hedgecock E.M. Cell. 1996; 85: 829-839Google Scholar). The expression of DDB2 is cell cycle-regulated, peaking at the G1/S boundary (17Nag A. Bondar T. Shiv S. Raychaudhuri P. Mol. Cell. Biol. 2001; 21: 6738-6747Google Scholar). DDB2, but not DDB1, mRNA and protein levels are induced 2- to 3-fold by UV irradiation (19Itoh T. Nichols A. Linn S. Oncogene. 2001; 20: 7041-7050Google Scholar). DDB1 binds to the apolipoprotein B (apoB) gene regulatory factor 2 (BRF-2), and the DDB1-BRF-2 heterodimer has been suggested to be required for optimum-specific apoB gene expression (20Krishnamoorthy R.R. Lee T.H. Butel J.S. Das H.K. Biochemistry. 1997; 36: 960-969Google Scholar). DDB1 also binds to viral transcriptional transactivators, including the hepatitis B virus X protein (HBVx) (21Butel J.S. Lee T.H. Slagle B.L. Princess Takamatsu Symp. 1995; 25: 185-198Google Scholar) and the V proteins from paramyxovirus, simian parainfluenza virus 5, mumps virus, human parainfluenza virus 2, and measles virus (22Lin G.Y. Lamb R.A. J. Virol. 2000; 74: 9152-9166Google Scholar). DDB2 also has been shown recently to bind HBVx protein (23Nag A. Datta A. Yoo K. Bhattacharyya D. Chakrabortty A. Wang X. Slagle B.L. Costa R.H. Raychaudhuri P. J. Virol. 2001; 75: 10383-10392Google Scholar). Because nuclear levels of DDB increase in late G1, DDB has been proposed to participate in nuclear functions of HBVx during the late G1 phase of the cell cycle. Despite this information, the precise physiological role(s) of DDB still remain elusive. We have identified three domains that are highly conserved in DDB1 sequence homologues of Homo sapiens, Mus musculus, Drosophila melanogaster, C. elegans, Dictyostelium discoideum,Arabidopsis thaliana, S. pombe (24Zolezzi F. Linn S. Gene. 2000; 245: 151-159Google Scholar), andOryza sativa. 2Unpublished data. 2Unpublished data. Two other reports assigned the same S. pombe predicted polypeptide to the DDB1 family of proteins (25Mintz P.J. Patterson S.D. Neuwald A.F. Spahr C.S. Spector D.L. EMBO J. 1999; 18: 4308-4320Google Scholar, 26Neuwald A.F. Poleksic A. Nucleic Acids Res. 2000; 28: 3570-3580Google Scholar). The overall identity between the human and S. pombe (previously published as NCBI accession number SPAC17H9.10c) sequences is 26%; the identities in domains 1, 2, and 3 are 45, 40, and 21%, respectively. To our knowledge, no sequence homologue of DDB1 has been found in Saccharomyces cerevisiae. S. pombe has been used extensively to study DNA repair and cell cycle control (27Humphrey T. Mutat. Res. 2000; 451: 211-226Google Scholar, 28McCready S.J. Osman F. Yasui A. Mutat. Res. 2000; 451: 197-210Google Scholar). S. pombe appears to be more closely related to animal cells with respect to cell cycle regulation and chromosome structure and segregation than is S. cerevisiae (29Zhao Y. Lieberman H.B. DNA Cell Biol. 1995; 14: 359-371Google Scholar) from which it diverged 330–420 million years ago (30Sipiczki M. Genome Biol. 2000; 1 (Reviews): 1011.1-1011.4Google Scholar). In this study we report the cloning of the S. pombesequence homologue of the human DDB1 gene,ddb1+, and the characterization of theddb1 deletion mutant. ddb1+ is not an essential gene, and the ddb1Δ cells show an elongated phenotype and nuclear abnormalities. The sensitivity of theddb1 deletion strain to a variety of DNA-damaging agents, to hydroxyurea, and to thiabendazole was compared with those of the null strains rad13 (the gene encoding for the homologue of the human XPG protein (31Edwards R.J. Carr A.M. Methods Enzymol. 1997; 283: 471-494Google Scholar)), rhp51 (the gene encoding for the homologue of the human RAD51 protein (32Muris D.F. Vreeken K. Carr A.M. Broughton B.C. Lehmann A.R. Lohman P.H. Pastink A. Nucleic Acids Res. 1993; 21: 4586-4591Google Scholar)), and cds1 (a gene implicated in the intra-S checkpoint response, the homologue of the human CHEK2 (33Boddy M.N. Furnari B. Mondesert O. Russell P. Science. 1998; 280: 909-912Google Scholar, 34Lindsay H.D. Griffiths D.J. Edwards R.J. Christensen P.U. Murray J.M. Osman F. Walworth N. Carr A.M. Genes Dev. 1998; 12: 382-395Google Scholar)). DISCUSSIONDDB from mammalian cells is a heterodimer of DDB1 and DDB2 (2Keeney S. Chang G.J. Linn S. J. Biol. Chem. 1993; 268: 21293-212300Google Scholar, 3Reardon J.T. Nichols A.F. Keeney S. Smith C.A. Taylor J.S. Linn S. Sancar A. J. Biol. Chem. 1993; 268: 21301-21308Google Scholar, 4Fujiwara Y. Masutani C. Mizukoshi T. Kondo J. Hanaoka F. Iwai S. J. Biol. Chem. 1999; 274: 20027-20033Google Scholar). As noted in the Introduction, DDB1 is an evolutionarily conserved protein; sequence homologues have been identified in mammals, worms, insects and plants. DDB2, however, is less conserved, and DDB2 sequence homologues have been identified only in mammals and plants (24Zolezzi F. Linn S. Gene. 2000; 245: 151-159Google Scholar). 3Unpublished data. Members of the DDB1 family do not contain nuclear localization signals, whereas all of the DDB2 homologues contain multiple nuclear localization signals (1Dualan R. Brody T. Keeney S. Nichols A.F. Admon A. Linn S. Genomics. 1995; 29: 62-69Google Scholar). 4Unpublished data. Nevertheless, when the Ddb1p-Myc fusion protein was expressed under the control of the natural chromosomal promoter, an almost exclusivel" @default.
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