Targeted disruption of Np95 gene renders murine embryonic stem cells hypersensitive to DNA damaging agents and DNA replication blocks.
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.
Muto
Masahiro
M
Research Center for Radiation Safety, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba-shi, Chiba 263-8555, Japan.
Kanari
Yasuyoshi
Y
Kubo
Eiko
E
Takabe
Tamami
T
Kurihara
Takayuki
T
Fujimori
Akira
A
Tatsumi
Kouichi
K
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000096:biosynthesis; D004249:DNA Damage; D004261:DNA Replication / Q000187:drug effects*; D004622:Embryo, Mammalian / Q000166:cytology*; D006918:Hydroxyurea / Q000494:pharmacology; D008769:Methylnitronitrosoguanidine; D051379:Mice; D008969:Molecular Sequence Data; D012854:Sister Chromatid Exchange; D013234:Stem Cells / Q000187:drug effects*; D014466:Ultraviolet Rays
D008769:Methylnitronitrosoguanidine; D004247:DNA; D006918:Hydroxyurea