Schizosaccharomyces pombe Ddb1 Is functionally Linked to the Replication Checkpoint Pathway

Abstract

Schizosaccharomyces pombe Ddb1 is homologous to the mammalian DDB1 protein, which has been implicated in damaged-DNA recognition and global genomic repair. However, a recent study suggested that the S. pombe Ddb1 is involved in cell division and chromosomal segregation. Here, we provide evidence that the S. pombe Ddb1 is functionally linked to the replication checkpoint control gene cds1. We show that the S. pombe strain lacking ddb1 has slow growth due to delayed replication progression. Flow cytometric analysis shows an extensive heterogeneity in DNA content. Furthermore, the Δddb1 strain is hypersensitive to UV irradiation in S phase and is unable to tolerate a prolonged replication block imposed by hydroxyurea. Interestingly, the Δddb1 strain exhibits a high level of the Cds1 kinase activity during passage through S phase. Moreover, mutation of the cds1 gene relieves the defects observed in Δddb1 strain. The results suggest that many of the defects observed in Δddb1 cells are linked to an aberrant activation of Cds1, and that Ddb1 is functionally linked to Cds1. Schizosaccharomyces pombe Ddb1 is homologous to the mammalian DDB1 protein, which has been implicated in damaged-DNA recognition and global genomic repair. However, a recent study suggested that the S. pombe Ddb1 is involved in cell division and chromosomal segregation. Here, we provide evidence that the S. pombe Ddb1 is functionally linked to the replication checkpoint control gene cds1. We show that the S. pombe strain lacking ddb1 has slow growth due to delayed replication progression. Flow cytometric analysis shows an extensive heterogeneity in DNA content. Furthermore, the Δddb1 strain is hypersensitive to UV irradiation in S phase and is unable to tolerate a prolonged replication block imposed by hydroxyurea. Interestingly, the Δddb1 strain exhibits a high level of the Cds1 kinase activity during passage through S phase. Moreover, mutation of the cds1 gene relieves the defects observed in Δddb1 strain. The results suggest that many of the defects observed in Δddb1 cells are linked to an aberrant activation of Cds1, and that Ddb1 is functionally linked to Cds1. The UV-damaged DNA-binding protein DDB in mammals consists of two subunits, DDB1 and DDB2 (1Hwang B.J. Chu G. Biochemistry. 1993; 32: 1657-1666Crossref PubMed Scopus (94) Google Scholar). DDB has high affinity for UV-induced DNA lesions, cisplatin-modified DNA, and bent DNA (2Abramic M. Levine A.S. Protic M J. Biol. Chem. 1991; 266: 22439-22500Abstract Full Text PDF Google Scholar, 3Mizukoshi T Fujiwara Y. Iwai S. Nucleic Acids Symp. Ser. 1999; 42: 265-266Crossref Scopus (1) Google Scholar). A damage sensor function of DDB has been proposed (4Otrin V.R. Kuraoka I. Nardo T. McLenigan M. Eker A.P.M. Stefanini M. Levine A.S. Wood R.D. Mol. Cell Biol. 1998; 18: 3182-3190Crossref PubMed Google Scholar). A number of studies indicated a role for DDB in nucleotide excision repair; however, DDB plays a stimulatory but nonessential role in that process (1Hwang B.J. Chu G. Biochemistry. 1993; 32: 1657-1666Crossref PubMed Scopus (94) Google Scholar, 2Abramic M. Levine A.S. Protic M J. Biol. Chem. 1991; 266: 22439-22500Abstract Full Text PDF Google Scholar, 5Chu G. Chang E. Science. 1988; 242: 564-567Crossref PubMed Scopus (351) Google Scholar). The DDB2 gene is mutated in a subset of patients with xeroderma pigmentosum (XP-E), a hereditary disease manifested by sun sensitivity and high susceptibility to skin cancer (4Otrin V.R. Kuraoka I. Nardo T. McLenigan M. Eker A.P.M. Stefanini M. Levine A.S. Wood R.D. Mol. Cell Biol. 1998; 18: 3182-3190Crossref PubMed Google Scholar). No mutation in the DDB1 gene has been identified in XP patients. The mammalian DDB1 has been also implicated in other pathways. For example, it was shown to participate in E2F1-activated transcription (6Kazantsev A. Mu D. Nichol A.F. Zhao X. Linn S. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5014-5018Crossref PubMed Scopus (63) Google Scholar, 7Shiyanov P. Hayes S.A. Donepudi M. Nichols A.F. Linn S. Slagle B.L. Raychaudhuri P. Mol. Cell. Biol. 1999; 19: 4935-4943Crossref PubMed Scopus (85) Google Scholar). DDB1 is important in the life cycle of the hepatitis B virus and paramyxovirus (8Krishnamoorthy R.R. Lee T.H. Butel J.S. Das H.K. Biochemistry. 1997; 36: 960-969Crossref PubMed Scopus (25) Google Scholar, 9Lin G.Y. Lamb R.A. J. Virol. 2000; 74: 9152-9166Crossref PubMed Scopus (96) Google Scholar, 10Lin G.Y. Paterson R.G. Richardson C.D. Lamb R.A. Virology. 1998; 249: 189-200Crossref PubMed Scopus (118) Google Scholar, 11Martinho R.G Lindsay H.D Flaggs G. DeMaggio A.J. Hoekstra M.F. Carr A.M. Bentley N.J. EMBO J. 1998; 17: 7239-7249Crossref PubMed Scopus (134) Google Scholar). It was shown that the HBx protein encoded by hepatitis B virus and the V protein encoded by the paramyxoviruses associate with DDB1 and that these associations are critical for establishment of infection. Interestingly, DDB1, but not DDB2, is conserved in lower eukaryotes, including flies, worms, fission yeast, and slime mold (12Zolezzi F. Linn S. Gene (Amst.). 2000; 245: 151-159Crossref PubMed Scopus (28) Google Scholar). The Schizosaccharomyces pombe and human DDB1 genes share 46% sequence homology, with three domains of 66, 58, and 35% homology that are conserved among other species (12Zolezzi F. Linn S. Gene (Amst.). 2000; 245: 151-159Crossref PubMed Scopus (28) Google Scholar). Markov's model analysis of DDB1 structure predicts a number of β-propeller blades comprised of WD-like repeats (13Neuwald A.F. Poleksic A. Nucleic Acids Res. 2000; 28: 3570-3580Crossref PubMed Google Scholar). The position and number of these repeats are remarkably conserved in S. pombe Ddb1 (15 repeats in yeast versus 17 in humans), suggesting that the overall structure and function of the two proteins might be similar. Recently, Zolezzi et al. (14Zolezzi F. Fuss J. Uzawa S. Linn S. J. Biol. Chem. 2002; 277: 41183-41191Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) isolated a cDNA clone of the putative S. pombe orthologue of mammalian DDB1 and characterized the phenotype of an S. pombe strain deficient for ddb1. Their Δddb1 strain, although viable, displays an elongated cell phenotype, abnormal nuclear morphology, and chromosome segregation defects. Interestingly, that study failed to detect any significant involvement of ddb1 in nucleotide excision repair and suggested that the function of yeast Ddb1 is linked to chromosome segregation and cell division. However, a link between ddb1 and the DNA damage checkpoint pathways was not investigated. Several DNA damage checkpoint genes have been characterized in S. pombe. The rad3 gene encodes a member of the phosphatidylinositol 3-kinase family and activates distinct checkpoint pathways mediated by the Chk1 and the Cds1 kinases during DNA damage and replication stress, respectively (for review, see Refs. 15Rhind N. Russell P. J. Cell Sci. 2000; 113: 3889-3896Crossref PubMed Google Scholar and 16Wahl G.M. Carr A.M. Nat. Cell Biol. 2001; 3: 277-286Crossref PubMed Scopus (326) Google Scholar). Cds1 and Chk1 inhibit Cdc2 by inhibiting the activator phosphatase Cdc25 (17Furnari B. Blasina A. Boddy M.N. McGowan C.H. Russell P. Mol. Biol. Cell. 1999; 10: 833-845Crossref PubMed Scopus (178) Google Scholar, 18Zeng Y. Forbes K.C. Wu Z. Moreno S. Piwnica-Worms H. Enoch T. Nature. 1998; 395: 507-510Crossref PubMed Scopus (306) Google Scholar) and/or by activating the inhibitory kinases (19Boddy M.N. Furnari B. Mondesert O. Russell P. Science. 1998; 280: 909-912Crossref PubMed Scopus (279) Google Scholar, 20O'Connell M.J. Raleigh J.M. Verkade H.M. Nurse P. EMBO J. 1997; 16: 545-554Crossref PubMed Scopus (326) Google Scholar). By inhibiting Cdc2, these checkpoint kinases block cell cycle progression. Cds1 also is involved in recovery from replication stress (21Boddy M.N. Gaillard P.H. McDonald W.H. Shanahan P. Yates III, J.R. Russell P. Cell. 2001; 107: 537-548Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, 22Boddy M.N. Lopez-Girona A. Shanahan P. Interthal H. Heyer W.D. Russell P. Mol. Cell. Biol. 2000; 20: 8758-8766Crossref PubMed Scopus (242) Google Scholar, 23Murakami H. Okayama H. Nature. 1995; 374: 817-819Crossref PubMed Scopus (250) Google Scholar, 24Kim S.M. Huberman J.A. EMBO J. 2001; 20: 6115-6126Crossref PubMed Scopus (113) Google Scholar, 25Sogo J.M. Lopes M. Foiani M. Science. 2002; 297: 599-602Crossref PubMed Scopus (678) Google Scholar). S. pombe cells lacking Cds1 (Δcds1) very rapidly lose viability in the presence of hydroxyurea (HU) 1The abbreviations used are: HU, hydroxyurea; EMM, Edinburgh minimal media; HA, hemagglutinin; GST, glutathione S-transferase; MOPS, 4-morpholinepropanesulfonic acid; rDNA, ribosomal DNA; DAPI, 4,6-diamidino-2-phenylindole; FACS, fluorescence-activated cell sorter. despite being able to delay mitosis until replication is complete (23Murakami H. Okayama H. Nature. 1995; 374: 817-819Crossref PubMed Scopus (250) Google Scholar). It has been proposed that Cds1 can somehow stabilize stalled forks, preventing their collapse or regression. Analysis of the replication intermediates in HU-treated Cds1 mutant by two-dimensional gel electrophoresis revealed an increased X-line signal, reflecting a higher level of unresolved recombination intermediates (24Kim S.M. Huberman J.A. EMBO J. 2001; 20: 6115-6126Crossref PubMed Scopus (113) Google Scholar). Aberrant DNA structures were observed by electron microscopy in HU-treated S. cerevisiae cells when Rad53, the Cds1 homologue, was inactivated (25Sogo J.M. Lopes M. Foiani M. Science. 2002; 297: 599-602Crossref PubMed Scopus (678) Google Scholar). In response to replication stress caused by either depletion of dNTPs or DNA lesions, Cds1 is activated by phosphorylation in a Rad3-dependent manner (26Lindsay 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: 382-395Crossref PubMed Scopus (328) Google Scholar). The Rad3-mediated activation of Cds1 requires replication and other checkpoint Rad proteins (26Lindsay 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: 382-395Crossref PubMed Scopus (328) Google Scholar); however, the details of the activation process are not clear. It is possible that the signal “sensed” by the Cds1 activation pathway is a stalled replication fork (27Marchetti M.A. Kumar S. Hartsuiker E. Maftahi M. Carr A.M. Freyer G.A. Burhans W.C. Huberman J.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7472-7477Crossref PubMed Scopus (63) Google Scholar). In agreement with this hypothesis, replication initiation proteins Orp1 and Cdc18 are indispensable for Cds1 activation in response to HU (28Murakami H Yanow S.K. Griffiths D. Nakanishi M. Nurse P. Nat. Cell Biol. 2002; 4: 384-388Crossref PubMed Scopus (24) Google Scholar). Hsk1, a protein involved in origin firing and in post-initiation S phase events, is required also for induction of Cds1 activity (29Takeda T. H. H. Mol. Biol. Cell. 2001; 12: PubMed Scopus Google Scholar). The activation of Cds1 is to S although the and Cds1 proteins are the cell The S protein is an protein required for the activation of Cds1 and of the signal to Cds1 A.J. J. F. Carr A.M. Nat. Cell Biol. 2001; 3: PubMed Scopus Google Scholar, Russell P. Nat. Cell Biol. 2001; 3: PubMed Scopus Google Scholar). The studies suggest a role of ddb1 in the activation of Cds1. and were in yeast or in Edinburgh minimal with were and by a to for or were mutant were by or analysis by were by genomic Cds1, the of Cds1 was by and an by P. a The was and by and of Δddb1 Δddb1 strain, and and of the ddb1 were genomic DNA and used were and and and The was of the with and and S. pombe strain were the was by genomic and The of was to were in the genes with the the of the were by with in a in the presence of were by and in and The cells were with two of and by The was to a and the were with The were with the and were by and with in of the for The was by and the was to in a in the presence of The DNA was by of the intermediates were with and with The was in gel for with was and the in the and was the until the were The was for in DNA was to the by and by in a The were by of the genomic DNA with the The was and to the the The was to the was by in and and with a UV sensitivity of cells were and with a of For hydroxyurea cells were in hydroxyurea for and were were for for for is a of observed the an of two and were for and with Cell were in and by with for were with the the was by for and the was for high and The was in the and the were for in the was by the were in and were to with in the was with of T. by The signal was by Cds1 and were with of to of protein for with The were with of and with of kinase The kinase was for with of protein, of and in of the kinase were by in and were by by In of the was by a control of Flow and were in of cell for was B. T. E. 1997; PubMed Scopus Google Scholar) with the cells were in in the of and for The were by in of of and for were with of for with of and with A for were by microscopy to the signal and DNA was by the and the were For cells were in a with of in DAPI, and with a were the were by and can study in a in Δddb1 the function of we an S. pombe strain in which the complete of the ddb1 gene was by the gene by homologous The was by and DNA not Δddb1 strain to the strain by Zolezzi et al. (14Zolezzi F. Fuss J. Uzawa S. Linn S. J. Biol. Chem. 2002; 277: 41183-41191Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Zolezzi et al. (14Zolezzi F. Fuss J. Uzawa S. Linn S. J. Biol. Chem. 2002; 277: 41183-41191Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) that the cell of the Δddb1 mutant was the In to an phenotype, we that Δddb1 cells by increased for Δddb1 versus for the in and by the see of cds1 the Δddb1 growth or cells were and for DNA of the or by analysis of phase revealed a of DNA in Δddb1 cells The heterogeneity is to be caused by in cell the analysis was cell and which signal B. T. E. 1997; PubMed Scopus Google Scholar). it be due in to chromosomal segregation in the Δddb1 strain, by Zolezzi et al. (14Zolezzi F. Fuss J. Uzawa S. Linn S. J. Biol. Chem. 2002; 277: 41183-41191Abstract Full Text Full Text PDF PubMed Scopus (31) Google an aberrant S phase also the DNA A number of cells DNA between and which the of the cell cycle delay in Δddb1 we Δddb1 strain. The a block in were for and a to through the cell cycle was by and not DNA with the in mitosis delay in Δddb1 cells to the cells the delay in the Δddb1 cells with S to M suggesting either or S phase in the Δddb1 the slow growth phenotype of the Δddb1 strain was due to a in DNA we for the replication by two gel electrophoresis and a of Δddb1 we a strain a of in the Δddb1 cells with the were by to the the cells were by to the through mitosis and were in by and of the not the the cells were for analysis of the replication DNA isolated from and cells were to two-dimensional gel electrophoresis to the replication intermediates from the DNA by and The were with two an was used to the ribosomal and the was used to detect the (24Kim S.M. Huberman J.A. EMBO J. 2001; 20: 6115-6126Crossref PubMed Scopus (113) Google Scholar). The with the revealed of DNA and the In the replication of the and the signal to the In the replication intermediates isolated from Δddb1 cells were from to with a replication of the required in the Δddb1 cells with in the A was with the that from the In cells replication intermediates to the were and the to in the Δddb1 replication and the intermediates were the replication intermediates from and were for a in the Δddb1 cells with cells It is that this in replication is for the phenotype of the Δddb1 S. pombe ddb1 to UV in S Δddb1 cells are to UV irradiation F. Fuss J. Uzawa S. Linn S. J. Biol. Chem. 2002; 277: 41183-41191Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar and However, we observed that when by the Δddb1 strain not any significant in sensitivity to UV irradiation with the strain between and cells suggested the that UV sensitivity in the might be this we the of the and the cell cycle progression. The cells were by a to and the cells were and In cell cycle was by and of the and Δddb1 cells were in S phase to Interestingly, although the strain was to UV irradiation in S the ddb1 cells to UV irradiation in S phase. Δddb1 cells were to UV during and M with the and were the of S phase. cell cycle analysis not between cells in and S we not a that Δddb1 cells are hypersensitive to UV in phase. we UV sensitivity of the and Δddb1 cells in by for No significant in sensitivity to was observed not ddb1 for a with cells in S phase. S. pombe cells a in S phase with HU, and We observed that the Δddb1 cells to HU not the of the in viability of the Δddb1 cells in the presence of HU, we the and the Δddb1 cells with HU for in and the cells in were with DAPI, and nuclear was The of the Δddb1 strain was during the of and not Δddb1 cells to the replication checkpoint function and However, Δddb1 cell viability during the in of viability by a in the of Δddb1 cells by this The was by analysis and with HU and Δddb1 cells with DNA the of with the Δddb1 cells a for DNA with the strain. Moreover, was in the HU block and DNA of HU in DNA from the to in However, the DNA increased and by in the Δddb1 and/or segregation of nuclear cells during of the cells from the revealed that with and DNA and cells of aberrant mitosis comprised of the Δddb1 cells this these that Δddb1 cells are deficient in a prolonged replication stress by aberrant DNA replication to damage in the of ddb1. Δddb1 the for aberrant S phase and S of the Δddb1 strain or stress, we to the activity of the S phase checkpoint kinase Cds1. from the and the Δddb1 cells were to a and a The was with a higher of in The and the were with The were to kinase and protein We observed that the Cds1 activity was in the of the Δddb1 cells Cds1 is an S we Cds1 activity in the of cells through the cell The cell cycle from the block was by the cells and by for the protein, a for S phase the indicated were for Cds1 kinase and The Cds1 kinase activity in the and Δddb1 cells was the the to the A of Cds1 kinase activity in Δddb1 cells but not in cells S phase it that the of Ddb1 caused a replication stress, to the activation of Cds1 in this strain. of cds1 the in Δddb1 study the role of the replication checkpoint activation in Δddb1 we of ddb1 and the checkpoint kinase cds1. we observed complete of the Δddb1 defects in the strain. the mutant of a to the or the that the growth was increased with the Δddb1 strain analysis revealed that the putative S of the cells with of the Δddb1 strain was by of cds1 is with the role of Cds1 in the of S phase and that Cds1 is activated in Δddb1 We sensitivity to UV irradiation of the cells to that of Δddb1 and of cds1 the sensitivity of ddb1 mutant to the UV irradiation that the mutant was not to UV either of the we that these two proteins are involved in the pathway during UV (26Lindsay 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: 382-395Crossref PubMed Scopus (328) Google Scholar) and Δddb1 are deficient in recovery from replication stress, we Ddb1 and Cds1 function in the pathway replication stress imposed by HU The strain was to in HU the Δddb1 strain However, the of Δddb1 not sensitivity in the suggesting that Ddb1 and Cds1 function in the Δddb1 to Cds1 of cds1 defects in the Δddb1 we that Cds1 activity might be in the Δddb1 we the of Cds1 Δddb1 A from a was in the Δddb1 or strain. the Δddb1 strain and growth the was by the of cells very and significant growth delay these and not Cds1 was Δddb1 cells unable to growth of the cells was was observed with of The results a between Ddb1 and the replication checkpoint pathway in S. pombe. Moreover, the results also provide an the function of ddb1 in S. pombe. The presence of activated Cds1 in Δddb1 cells that Ddb1 or activation of Cds1. We not see a between Cds1 and Ddb1 not we it is an The replication checkpoint protein Cds1 is activated by stalled replication and by that replication the high level of activated Cds1 in Δddb1 cells during the passage through S phase a role of Ddb1 in of stalled replication or replication stress during Cds1 is to a role during replication stress by a cell cycle S phase (26Lindsay 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: 382-395Crossref PubMed Scopus (328) Google Scholar). In the Δddb1 strain, the in S phase is suggesting that the function of Cds1 during replication stress The of recovery from prolonged replication stress is not however, it has been proposed that of replication is required for In the of replication or to structures (24Kim S.M. Huberman J.A. EMBO J. 2001; 20: 6115-6126Crossref PubMed Scopus (113) Google Scholar, 25Sogo J.M. Lopes M. Foiani M. Science. 2002; 297: 599-602Crossref PubMed Scopus (678) Google Scholar). it is possible that Ddb1 is involved in of replication and that Δddb1 cells are in the replication a prolonged replication defects can in abnormal For example, in a when two replication of DNA in between the be it is that the aberrant mitosis observed in a of the Δddb1 cells of in HU was a of abnormal The were observed a lower in the Δddb1 strain any F. Fuss J. Uzawa S. Linn S. J. Biol. Chem. 2002; 277: 41183-41191Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar and which be a of a replication it is possible that HU the defects observed in the Δddb1 strain. A role for Ddb1 in DNA replication the that the Δddb1 strain is hypersensitive to UV irradiation during S phase. be that the Δddb1 strain is deficient in nucleotide excision repair. In this is by but in S phase for recombination is for in increased sensitivity to DNA the other this model higher sensitivity to irradiation during which was not model of the S phase to UV is replication UV-induced and the of replication is in the Δddb1 model is it is with the sensitivity of the Δddb1 strain to (14Zolezzi F. Fuss J. Uzawa S. Linn S. J. Biol. Chem. 2002; 277: 41183-41191Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) and the S to M in these cells for model Ddb1 in replication is by the of Cds1 and We that of cds1 of the Δddb1 strain, suggesting that the defects in Δddb1 cells were caused by Cds1. we failed to detect a between the Ddb1 and Cds1 we that the of Cds1 proteins and the are important in the between Cds1 and We in the of the Cds1 in an to the in the Δddb1 strain. It is that mutation in cds1 has been shown to defects observed in with in the replication and and Hsk1, the two of the replication initiation are for origin firing and have an function in the phase are required for recovery from replication stress (29Takeda T. H. H. Mol. Biol. Cell. 2001; 12: PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar, Mol. Cell. Biol. 2000; 20: PubMed Scopus Google Scholar). The phenotype of an mutant is by of cds1 Mol. Cell. Biol. 2000; 20: PubMed Scopus Google Scholar). Moreover, sensitivity to in a strain is by a cds1 J. S. Mol. Cell. Biol. 2002; PubMed Scopus Google Scholar). these with replication we suggest that Ddb1 plays a role in the of the replication in a that is linked to Cds1. We for Russell for and Cds1 and for Cds1