Targeted disruption of Np95 gene renders murine embryonic stem cells hypersensitive to DNA damaging agents and DNA replication blocks

Masahiro Muto, Yasuyoshi Kanari, Eiko Kubo, Tamami Takabe, Takayuki Kurihara, Akira Fujimori, Kouichi Tatsumi

Research output: Contribution to journalArticlepeer-review

85 Citations (Scopus)

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 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.

Original languageEnglish
Pages (from-to)34549-34555
Number of pages7
JournalJournal of Biological Chemistry
Volume277
Issue number37
DOIs
Publication statusPublished - 2002 Sep 13

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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