TY - JOUR
T1 - Targeted disruption of Np95 gene renders murine embryonic stem cells hypersensitive to DNA damaging agents and DNA replication blocks
AU - Muto, Masahiro
AU - Kanari, Yasuyoshi
AU - Kubo, Eiko
AU - Takabe, Tamami
AU - Kurihara, Takayuki
AU - Fujimori, Akira
AU - Tatsumi, Kouichi
PY - 2002/9/13
Y1 - 2002/9/13
N2 - 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 the Np95 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 transfected Np95 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 for Np95-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.
AB - 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 the Np95 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 transfected Np95 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 for Np95-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.
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U2 - 10.1074/jbc.M205189200
DO - 10.1074/jbc.M205189200
M3 - Article
C2 - 12084726
AN - SCOPUS:0037072831
VL - 277
SP - 34549
EP - 34555
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 37
ER -