25. Targeted Disruption of Np95 Gene Renders Murine ES Cells Hypersensitive to DNA Damaging Agents and DNA Replication Blocks
Masahiro Muto, Yasuyoshi Kanari1, Eiko Kubo, Tamami Takabe, Takayuki Kurihara2, Akira Fujimori and Kouichi Tatsumi
(1Kinki Univ. School of Medicine; 2Medical Research Institute, Kanazawa Medical Univ.)
Keywords: X-rays, UV, MNNG, SCE, cell cycle, hydroxyurea, replication fork
NP95, which contains a ubiquitin-like domain, a cyclin A/E-Cdk2 phosphorylation site, Rb binding motif and ring finger domain, has been shown to be colocalized as foci with PCNA in early and mid-S phase nuclei. We established Np95 nulligous ES 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, ultraviolet light (UV), N-methyl-N''-nitro-N-nitrosoguanidine (MNNG) and hydroxyurea (HU) than ES 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 HU concurrently to a level similar to that of Np95+/- cells, though slightly below that of wild type (Np95+/+) cells. These findings suggested that NP95 played a role in the repair process for DNA damages 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 inferred that NP95 functioned as a common component in the multiple response pathways against DNA damages and replication arrest and thereby contributed to the genomic stability.
Publications:
1) Muto, M., Kanari, Y., Kubo, E., Takabe, T., Kurihara, T., Fujimori, A., and Tatsumi, K.: J. Biol. Chem.(in press. Published online June 25, 2002., 10.1074/jbc.M205189200).
2) Miura, M., Watanabe, H., Sasaki, T., Tatsumi, K.,Muto, M.: Exp Cell Res., 263, 202-208, 2001.
3) Uemura, T., Kubo, E., Kanari, Y., Ikemura, T., Tatsumi, K., Muto, M.: Cell Struct. Funct., 25, 149-159, 2000.
4) Fujimori, A., Matsuda, Y., Takemoto, Y., Hashimoto, Y., Kubo, E., Araki, R., Fukumura, R., Mita, K., Tatsumi, K., Muto, M.: Mamm. Genome, 9, 1032-1035, 1998.