Targeted Disruption of Np95 Gene Renders Murine Embryonic Stem Cells Hypersensitive to DNA Damaging Agents and DNA Replication Blocks

Abstract

NP95, which contains a ubiquitin-like domain, a cyclin A/E-Cdk2 phosphorylation site, a retinoblastoma (Rb) binding motif, and a ring finger domain, has been shown to be colocalized as foci with proliferating cell nuclear antigen in early and mid-S phase nuclei. We established Np95 nulligous embryonic stem cells by replacing the exons 2–7 of theNp95 gene with a neo cassette and by selecting out a spontaneously occurring homologous chromosome crossing over with a higher concentration of neomycin. Np95-null cells were more sensitive to x-rays, UV light,N-methyl-N′′-nitro-N-nitrosoguanidine (MNNG), and hydroxyurea than embryonic stem wild type (Np95+/+) or heterozygously inactivated (Np95+/−) cells. Expression of transfectedNp95 cDNA in Np95-null cells restored the resistance to x-rays, UV, MNNG, or hydroxyurea concurrently to a level similar to that of Np95+/− cells, although slightly below that of wild type (Np95+/+) cells. These findings suggest that NP95 plays a role in the repair of DNA damage incurred by these agents. The frequency of spontaneous sister chromatid exchange was significantly higher forNp95-null cells than for Np95+/+cells or Np95+/− cells (p < 0.001). We conclude that NP95 functions as a common component in the multiple response pathways against DNA damage and replication arrest and thereby contributes to genomic stability. NP95, which contains a ubiquitin-like domain, a cyclin A/E-Cdk2 phosphorylation site, a retinoblastoma (Rb) binding motif, and a ring finger domain, has been shown to be colocalized as foci with proliferating cell nuclear antigen in early and mid-S phase nuclei. We established Np95 nulligous embryonic stem cells by replacing the exons 2–7 of theNp95 gene with a neo cassette and by selecting out a spontaneously occurring homologous chromosome crossing over with a higher concentration of neomycin. Np95-null cells were more sensitive to x-rays, UV light,N-methyl-N′′-nitro-N-nitrosoguanidine (MNNG), and hydroxyurea than embryonic stem wild type (Np95+/+) or heterozygously inactivated (Np95+/−) cells. Expression of transfectedNp95 cDNA in Np95-null cells restored the resistance to x-rays, UV, MNNG, or hydroxyurea concurrently to a level similar to that of Np95+/− cells, although slightly below that of wild type (Np95+/+) cells. These findings suggest that NP95 plays a role in the repair of DNA damage incurred by these agents. The frequency of spontaneous sister chromatid exchange was significantly higher forNp95-null cells than for Np95+/+cells or Np95+/− cells (p < 0.001). We conclude that NP95 functions as a common component in the multiple response pathways against DNA damage and replication arrest and thereby contributes to genomic stability. monoclonal antibody N-methyl-N′′-nitro-N-nitrosoguanidine embryonic stem cell proliferating cell nuclear antigen hydroxyurea E14 wild type sister chromatid exchange phosphate-buffered saline 0.1 0.1% Tween in PBS radioresistant DNA synthesis gray ionizing radiation methyl methanesulfonate ataxia-telangiectasia-mutated mean Entry into and progression through the mammalian cell cycle are highly regulated processes that involve a number of positively and negatively acting proteins at the molecular level. When growing cells are exposed to DNA damage or DNA replication blocks, they arrest their cell cycle processes, apparently to allow time for repair to be completed satisfactorily. This arrest is part of a “checkpoint” function that monitors the physical state of DNA at different stages of the cycle. Cells that have lost a checkpoint control may be as DNA damage-sensitive as cells that have lost DNA repair capability. Many genes are involved in controlling the cell cycle and determining checkpoints (1Elledge S.J. Science. 1996; 274: 1664-1672Crossref PubMed Scopus (1765) Google Scholar, 2Hartwell L.H. Weinert T.A. Science. 1989; 246: 629-634Crossref PubMed Scopus (2425) Google Scholar, 3Zhou B.S. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2637) Google Scholar). Furthermore, a possible link between checkpoint failure, hypersensitivity to DNA damage or replication blockage, and genomic instability has provided an important insight into processes contributing to the cellular dysfunction that leads to cancer (4Hartwell L.H. Kastan M.B. Science. 1994; 266: 1821-1828Crossref PubMed Scopus (2312) Google Scholar). We previously produced a monoclonal antibody, Th-10a mAb,1 that recognizes a 95-kDa mouse nuclear protein (NP95). NP95 was detected by the Th-10a mAb, specifically in the S phase of normal mouse thymocytes. In contrast, mouse T cell lymphoma cells showed a constantly high level for NP95 accumulation irrespective of cell stages during the cell cycle (5Muto M. Utsuyama M. Horiguchi T. Kubo E. Sado T. Hirokawa K. Cell Prolif. 1995; 28: 645-657Crossref PubMed Scopus (23) Google Scholar). By immunoscreening a λgt-11 cDNA expression library with the Th-10a mAb, we isolated the cDNA encoding NP95 (6Fujimori A. Matsuda Y. Takemoto Y. Hashimoto Y. Kubo E. Araki R. Fukumura R. Mita K. Tatsumi K. Muto M. Mamm. Genome. 1998; 9: 1032-1035Crossref PubMed Scopus (72) Google Scholar). Sequencing of the whole 3.5-kb cDNA revealed that NP95 is a novel nuclear protein with an open reading frame consisting of 782 amino acids. The open reading frame contains an unusual N-terminal domain that bears a striking resemblance to ubiqutin, a leucine zipper motif, a zinc finger motif, a potential ATP/GTP binding site, a putative cyclin A/E-cdk2 phosphorylation site, retinoblastoma protein (Rb)-binding motifs, and a ring finger domain (6Fujimori A. Matsuda Y. Takemoto Y. Hashimoto Y. Kubo E. Araki R. Fukumura R. Mita K. Tatsumi K. Muto M. Mamm. Genome. 1998; 9: 1032-1035Crossref PubMed Scopus (72) Google Scholar). NP95 was shown to be localized in S phase nuclei as dot-like foci (7Uemura T. Kubo E. Kanari Y. Ikemura T. Tatsumi K. Muto M. Cell Struct. Funct. 2000; 25: 149-159Crossref PubMed Scopus (48) Google Scholar). Double immunostaining for NP95 and chromatin-bound PCNA revealed that NP95 was almost exclusively colocalized with chromatin-bound PCNA throughout the nucleus in early S phase and partly in mid-S phase. However, distinct localization of the two proteins became evident in some part in mid-S phase and late-S phase (7Uemura T. Kubo E. Kanari Y. Ikemura T. Tatsumi K. Muto M. Cell Struct. Funct. 2000; 25: 149-159Crossref PubMed Scopus (48) Google Scholar, 8Miura M. Watanabe H. Sasaki T. Tatsumi K. Muto M. Exp. Cell Res. 2001; 263: 202-208Crossref PubMed Scopus (34) Google Scholar). These results suggested that NP95, rather than committing DNA replication itself as a component of replication machinery, contributes to some other DNA replication-linked events (8Miura M. Watanabe H. Sasaki T. Tatsumi K. Muto M. Exp. Cell Res. 2001; 263: 202-208Crossref PubMed Scopus (34) Google Scholar). In this study, to gain further insights into the physiological role of NP95, we attempted to produce a targeted disruption of mouseNp95. We made strenuous but ultimately futile efforts to obtain Np95 null mice, probably because Np95inactivation caused mid-gestational embryonic lethality. We, therefore, established Np95-null embryonic stem (ES) cells by treatingNp95+/− ES cells with a higher concentration of G418 (2 mg/ml) to select out a mitotic crossing over. We examined the effect of the homozygous Np95 disruption on the sensitivity to x-rays, UV light, andN-methyl-N′′-nitro-N-nitrosoguanidine (MNNG) and found that Np95-null ES cells were more sensitive to treatment with these agents than ES wild type (E14.wt) or 19(Np95+/−) cells. In addition,Np95-null ES cells turned out to be more sensitive to DNA replication inhibitor hydroxyurea (HU) than E14.wt or 19(Np95+/−) cells. To obtain further insights into the role of NP95 in DNA recombination and chromosomal stability, we examined the frequency of spontaneous sister chromatid exchange (SCE) in NP95-deficient cells. In view of the characteristics of NP95-deficient cells, we suggest that NP95 functions as a common component in the multiple DNA damage response pathways and that it plays a role in the maintenance of genomic stability. To construct the targeting vector of Np95, genomic DNA segments corresponding to the Np95 were obtained by screening of the strain 129 genomic library, λ129 (9Gomi H. Yokoyama T. Fujimoto K. Ikeda T. Katoh A. Itohara S. Neuron. 1995; 14: 29-41Abstract Full Text PDF PubMed Scopus (231) Google Scholar), with 1.8 kb of N-terminal cDNA of Np95 cDNA. Selected genomic fragments containing the Np95 gene were subcloned into pBluescriptII SK (−). Sequencing of each clone was done using dye terminator beginning with the T3 and T7 ends (GenBankTMaccession number AB066245). The exon-intron boundary predictions were based upon comparison of the genomic sequence with Np95cDNA (6Fujimori A. Matsuda Y. Takemoto Y. Hashimoto Y. Kubo E. Araki R. Fukumura R. Mita K. Tatsumi K. Muto M. Mamm. Genome. 1998; 9: 1032-1035Crossref PubMed Scopus (72) Google Scholar). The targeting vector, pNPKO1, was constructed using two genomic clones, pNP1 and pNP7. The upstream 1.5-kb BamHI fragment from pNP1 and downstream 6.2-kbSalI/EcoRI fragment from pNP7 were inserted into sites of restriction enzymes, ClaI and XbaI, in pgk-Neo cassette. This insertion resulted in deletion from exon 2 to exon 7 (see Fig. 1A). ES cells, E14, were kindly provided by Dr. Martin L. Hooper (Department of Pathology, University of Edinburgh Medical School, Edinburgh, Scotland, UK) (10Hooper M. Hardy K. Handyside A. Hunter S. Monk M. Nature. 1987; 326: 292-295Crossref PubMed Scopus (923) Google Scholar). These were cultured in ES medium containing Dulbecco’s modified Eagle’s medium (430-2800ED, Invitrogen) supplemented with 15% fetal calf serum,l-glutamine, penicillin-streptomycin, nucleoside mixture (adenosine, guanosine, thymidine, cytidine and uridine), non-essential amino acids (Invitrogen), 10−4m2-mercaptoethanol, and 103 S. A. Hooper A. S. Full Text PDF PubMed Scopus Google Scholar), and they were in a ES medium nucleoside was ES E14 cells were on embryonic cells, in the of of targeting vector, pNPKO1, and on cells with G418 at for 7 were by using in exon and in the of cassette. were with using and fragment of ES cell for the Np95 gene were for and at a of cultured for and in the of 2 of that for were by using A. of the Np95 gene was by with Th-10a ES cell for 7 were and for in The cells were and of these cells were and in of of cell was to of homologous using the exon and (−). was with as at consisting of for for and for with a at DNA isolated from ES cell was with by through and to in The is in Fig. and with The was in and with ES cells were in and they were with and in (8Miura M. Watanabe H. Sasaki T. Tatsumi K. Muto M. Exp. Cell Res. 2001; 263: 202-208Crossref PubMed Scopus (34) Google of whole cell were by and to in in PBS for were and with or antibody in in PBS for at with Tween in of antibody was revealed using antibody at in in PBS for at and with were by Cells were with for at with PBS supplemented with fetal calf with of monoclonal antibody in and for at The cells were with and in for at the cells were with and to Np95 cDNA was inserted into vector containing a and To construct expression vector, an Np95 cDNA fragment containing the sequence in the and with a and in the was inserted into the BamHI of the vector or vector were by with and cells were with these using to provided by the The cells were cultured for in the of to were and DNA from these was isolated and to the Np95 cDNA was detected by these were to produce NP95 by immunostaining of the cells with and ES were and on at a from 103 to at for ES cells were exposed to of was with a at at with of and ES were on and cultured at for ES cells were and with of Dulbecco’s The cells were with UV from at The was as with a UV ES were and on at 103 cells at for ES cells were exposed to of in ES medium for at with and cultured further in ES ES were and on at 103 cells. for at ES medium was and of were to the the treatment for at ES cells were with of Dulbecco’s modified Eagle’s medium and cultured further in ES these ES cells were with ES medium for 7 and were from to in of the of were based on at two or out in ES cells were into ES medium containing and were for of the each was further in ES medium for at 2 to from DNA were with of during the corresponding and cell were for with The were with and with of The were on by of of The were with with and and in a The of DNA synthesis in was as and as a of for as shown in ES were and on and and UV at a cell ES medium was and the cells were with x-rays, UV, and as The cells were cultured for two cell cycle with ES medium containing and with for the The cells were by and exposed to for at The cells were with the and in a of the The cell was and at for 2 chromatid was obtained by a S. Nature. PubMed Scopus Google Scholar, T. M. Tatsumi K. Res. 1987; Google Scholar). the cells on the were with in for with PBS and with The were exposed to at a of for 2 of the were in at for and with in for in and was for To further the physiological function of NP95, we cell with Np95 gene from the genomic library of strain 129 We constructed a targeting vector in which part of the Np95 genomic from exon 2 containing to exon 7 was by gene cassette with and in ES medium containing we isolated ES and two and were homologous in Np95 To obtain NP95-deficient ES cells in 19(Np95+/−) clone was in medium containing highly G418 (2 and were and were shown to be in of the and were and the Np95 gene was in these cells expression of NP95 is in S phase cells and with PCNA (7Uemura T. Kubo E. Kanari Y. Ikemura T. Tatsumi K. Muto M. Cell Struct. Funct. 2000; 25: 149-159Crossref PubMed Scopus (48) Google Scholar, 8Miura M. Watanabe H. Sasaki T. Tatsumi K. Muto M. Exp. Cell Res. 2001; 263: 202-208Crossref PubMed Scopus (34) Google Scholar), it was that NP95 was with DNA replication and that NP95-deficient ES cells were However, NP95-deficient ES cell were in and the time was similar to E14.wt and 19(Np95+/−) cell of NP95-deficient ES cells was This that NP95 in DNA replication-linked events other than DNA replication itself as a replication machinery, a that is with a (8Miura M. Watanabe H. Sasaki T. Tatsumi K. Muto M. Exp. Cell Res. 2001; 263: 202-208Crossref PubMed Scopus (34) Google Scholar). PCNA is to be with proteins involved in DNA DNA and cell cycle H. Science. PubMed Scopus Google Scholar, Y. M. Cell PubMed Google Scholar, PubMed Scopus Google Scholar, S. 1994; Full Text PDF PubMed Scopus Google Scholar, R. S. Nature. 1994; PubMed Scopus Google Scholar, M.B. 1989; Full Text PDF PubMed Google Scholar, Science. 1996; 274: PubMed Scopus Google Scholar, S. Nature. 1994; PubMed Scopus Google Scholar, T. S. M. A. 2001; PubMed Scopus Google Scholar). To deletion of the Np95 gene the DNA we examined the sensitivity of NP95-deficient ES cells to of DNA and NP95-deficient ES cells and were exposed to of x-rays, and the was obtained as shown in The results that NP95-deficient ES cells were more sensitive to than E14.wt cells or 19(Np95+/−) cells. to the of cells to was for cells, cells, and cells. NP95-deficient cells were more sensitive to UV and the MNNG, than E14.wt cells. for cells and E14.wt cells was for UV and and for MNNG, and NP95-deficient ES cells an to the different of DNA agents as with E14.wt cells. The in the between E14.wt cells and 19(Np95+/−) cells was for 2 and of but for and of of disruption and of on cellular resistance to UV and shown in as a function of UV in cells Np95 cells cells and cells were of a out in shown as a function of the concentration of in cells Np95 cells 19(Np95+/−) cells cells cells and cells were from two each out in NP95-deficient ES cells were shown to be more sensitive to DNA agents than ES wild type cells, we examined with Np95 cDNA into NP95-deficient ES cells the response to DNA damage to a level similar to that of E14.wt and 19(Np95+/−) cells. We constructed NP95 expression vector, in which Np95cDNA with a and at the was inserted into expression vector The NP95 expression construct was and into cells. screening of based on and we obtained a number of of cells. two and were to have an by and of NP95 protein and cells were to as a The with Np95 cDNA restored the resistance of cells to to a although similar to that of 19(Np95+/−) cells and in Fig. than to the wild type level of sensitivity were as the in the between and E14.wt was < for 2 < for of these cells against NP95 by the cell revealed that the of NP95 expression in and cells a of of E14.wt or 19(Np95+/−) These of NP95 expression in and may the The of NP95 function by the vector itself was out was in the of the hypersensitivity to between cells and cells in an to UV or in Np95 cells that the cellular resistance to agents was although the of was cells in Fig. and vector effect on the sensitivity of cells to UV or cells in Fig. and These results that Np95 is of the genes cellular resistance concurrently to ionizing radiation UV, and Expression of a in caused an sensitivity to methyl methanesulfonate and UV with an DNA replication and a of checkpoint control 1998; PubMed Scopus Google Scholar, S. A. 1998; PubMed Scopus Google Scholar). NP95-deficient cells were to x-rays, UV, and 2 and it possible to that the of NP95 results in an sensitivity to NP95-deficient ES cells were more sensitive to than E14.wt cells or 19(Np95+/−) cells cells and cells in Fig. cells restored the resistance to to an similar to that of 19(Np95+/−) cells cells and cells in Fig. cells and cells showed a similar hypersensitivity to as we theNp95 gene to the cellular to with DNA replication incurred by The cultured from the with are sensitive to but cells or in the sensitivity to UV, or of DNA These cells are by an S. A. PubMed Scopus Google Scholar, Res. PubMed Scopus Google Scholar, 1994; PubMed Scopus Google Scholar). the other the of a of in an sensitivity to UV light, and and the arrest to these cells an in the of an of wild type in cells the to DNA synthesis to a possible between and functions 1998; PubMed Scopus Google Scholar). NP95-deficient cells are sensitive to DNA agents and DNA replication we NP95-deficient ES cells the characteristics of an We examined the time of in DNA synthesis the of cells to and cells are with at The of a NP95-deficient ES cells and control ES cells is in Fig. In and cells, the replication a of that of the at treatment This was by a phase synthesis that by the level was of that occurring in The of DNA synthesis in and cells were almost the as of E14.wt and The results that NP95-deficient ES cells are to the DNA synthesis to x-rays, similar to the which a or wild type To the role of NP95 in chromosomal stability, we the spontaneous in NP95-deficient ES cells with in 19(Np95+/−) and E14.wt cells. the of spontaneous sister chromatid in and cells. The mean of for NP95-deficient cells and was significantly higher than that for E14.wt and 19(Np95+/−) cells (p < 0.001). The mean in 19(Np95+/−) cells was significantly different from that in wild type E14.wt cells The mean between cells and cells were significantly different (p In the study, to further the function of NP95, we established NP95-deficient ES cells by a neo cassette into exon 2 to exon 7 of the Np95 gene on and examined the cellular to different of DNA damage by x-rays, UV, and ES cells were found to be more sensitive to of these agents than or 19(Np95+/−) cells cells with Np95 cDNA revealed the of the Np95 gene to the resistance to x-rays, UV, or to almost similar as of 19(Np95+/−) cells, that at part of the normal response to to UV, or be to Np95 gene function 2 and The DNA repair is itself by a of genes that the cellular to of DNA Np95-null cells a similar hypersensitivity to these different of DNA it is that NP95 plays a common role in different repair Np95-null ES cells were to a to as with E14.wt or 19(Np95+/−) cells and results in the of the these of the DNA replication is and synthesis of fragments on the may to of DNA in to the replication The hypersensitivity to cells with the of resistance by the Np95 cDNA expression in the null cells that NP95 upon cells a to with at DNA has been shown that of or the to x-rays, UV light, and PubMed Scopus Google Scholar, Elledge S.J. 1998; PubMed Scopus Google and are for checkpoint to x-rays, UV light, and A. M. 1996; PubMed Scopus Google Scholar, T.A. L.H. 1994; PubMed Scopus Google Scholar). In to cell cycle checkpoint these genes have been in the of DNA repair T. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, Nature. 2001; PubMed Scopus Google Scholar). with the is the and mammalian and A. M. 1996; PubMed Scopus Google Scholar, S. A. 1996; PubMed Scopus Google Scholar), an expression of a of in has been shown to an sensitivity to UV, and and to a of checkpoint control 1998; PubMed Scopus Google Scholar, S. A. 1998; PubMed Scopus Google Scholar). S. S. K. S. A. Elledge S.J. 2000; 14: PubMed Scopus Google a in which and two in the mammalian DNA damage response that to different of DNA of wild type or was shown to the of in cells in that may be a between and 1998; PubMed Scopus Google Scholar). The DNA synthesis in and Np95-null ES cells an to was of Np95 it is to that the of Np95-null ES cells that of cells 1998; PubMed Scopus Google Scholar, S. A. 1998; PubMed Scopus Google Scholar, S. S. K. S. A. Elledge S.J. 2000; 14: PubMed Scopus Google Scholar, H. K. H. T. Ikeda K. K. M. K. 2000; 14: Google with to the to different of DNA damage and the DNA replication We have that NP95 is almost colocalized with chromatin-bound PCNA throughout the nuclei in early S phase and partly in mid-S phase. However, distinct localization of the two proteins in some part evident in mid-S phase and late-S phase (7Uemura T. Kubo E. Kanari Y. Ikemura T. Tatsumi K. Muto M. Cell Struct. Funct. 2000; 25: 149-159Crossref PubMed Scopus (48) Google Scholar, 8Miura M. Watanabe H. Sasaki T. Tatsumi K. Muto M. Exp. Cell Res. 2001; 263: 202-208Crossref PubMed Scopus (34) Google Scholar). during NP95 is in proliferating but in and that are proliferating (7Uemura T. Kubo E. Kanari Y. Ikemura T. Tatsumi K. Muto M. Cell Struct. Funct. 2000; 25: 149-159Crossref PubMed Scopus (48) Google Scholar). PCNA has been as a of a of proteins that with to a Y. Elledge S.J. 2000; 14: PubMed Scopus Google Scholar). the other has been found to in response to DNA or DNA and it is that is for the maintenance of genomic and for the control of homologous recombination in and cells R. K. 2000; PubMed Scopus Google Scholar, M. K. S. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, R. R. Elledge S.J. 2000; 14: PubMed Scopus Google Scholar, R. A. Y. T. Full Text Full Text PDF PubMed Scopus Google Scholar, Res. 2000; Google Scholar). the between NP95 and PCNA or other proteins to be we that NP95 functions as a component of the containing frequency is a of chromosomal stability, and spontaneous chromosomal instability has been with cancer S.J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). during and DNA it is that the replication repair L. 1996; PubMed Scopus Google Scholar). The by a that for incurred by DNA agents. may spontaneously as a of the of Np95-null cells are to be in some common DNA repair are to more in Np95-null cells than in E14.wt or 19(Np95+/−) cells. was therefore, to the for more in NP95-deficient cells. The of spontaneous in ES cells were significantly higher than of E14.wt and 19(Np95+/−) cells (p < The of with 2 of were significantly higher in Np95-null cells and for and were (10Hooper M. Hardy K. Handyside A. Hunter S. Monk M. Nature. 1987; 326: 292-295Crossref PubMed Scopus (923) Google and M.B. 1989; Full Text PDF PubMed Google Scholar), than in E14.wt cells Y. M. Cell PubMed Google and 19(Np95+/−) cells S. 1994; Full Text PDF PubMed Scopus Google although the by in Np95-null cells was has been shown that spontaneous are significantly in cells the that homologous recombination is of the for in cells E. Sasaki M. Y. M. S. PubMed Scopus Google Scholar). the other spontaneous are as to in cells from with K. The of Scholar), in mouse cells that L. M. M. K. PubMed Scopus Google or H. 1998; Full Text Full Text PDF PubMed Google Scholar), and in cells with in L.H. PubMed Scopus Google Scholar). or of genes encoding these proteins have been to to genomic the frequency of spontaneously chromosome in NP95-deficient cells to be it is highly that NP95 is for the maintenance of chromosomal stability. we that NP95 functions as a common component in the multiple response pathways for DNA damage and that it plays some role in the maintenance of genomic stability. We Dr. M. L. Hooper (Department of Pathology, University of Edinburgh Medical School, Edinburgh, Scotland, UK) for kindly with E14 ES cells. The provided by S. and M. and by are