Deletion of mouse rad9 causes abnormal cellular responses to DNA damage, genomic instability, and embryonic lethality.
The fission yeast Schizosaccharomyces pombe rad9 gene promotes cell survival through activation of cell cycle checkpoints induced by DNA damage. Mouse embryonic stem cells with a targeted deletion of Mrad9, the mouse ortholog of this gene, were created to evaluate its function in mammals. Mrad9(-/-) cells demonstrated a marked increase in spontaneous chromosome aberrations and HPRT mutations, indicating a role in the maintenance of genomic integrity. These cells were also extremely sensitive to UV light, gamma rays, and hydroxyurea, and heterozygotes were somewhat sensitive to the last two agents relative to Mrad9(+/+) controls. Mrad9(-/-) cells could initiate but not maintain gamma-ray-induced G(2) delay and retained the ability to delay DNA synthesis rapidly after UV irradiation, suggesting that checkpoint abnormalities contribute little to the radiosensitivity observed. Ectopic expression of Mrad9 or human HRAD9 complemented Mrad9(-/-) cell defects, indicating that the gene has radioresponse and genomic maintenance functions that are evolutionarily conserved. Mrad9(+/-) mice were generated, but heterozygous intercrosses failed to yield Mrad9(-/-) pups, since embryos died at midgestation. Furthermore, Mrad9(-/-) mouse embryo fibroblasts were not viable. These investigations establish Mrad9 as a key mammalian genetic element of pathways that regulate the cellular response to DNA damage, maintenance of genomic integrity, and proper embryonic development.
Center for Radiological Research, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA.
Kevin M
KM
Hopkins
Wojtek
W
Auerbach
Xiang Yuan
XY
Wang
M Prakash
MP
Hande
Haiying
H
Hang
Debra J
DJ
Wolgemuth
Alexandra L
AL
Joyner
Howard B
HB
Lieberman
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D002869:Chromosome Aberrations; D004249:DNA Damage; D004622:Embryo, Mammalian / Q000033:anatomy & histology / Q000502:physiology*; D017353:Gene Deletion; D005786:Gene Expression Regulation; D018816:Genes, cdc; D042822:Genomic Instability; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D020287:In Situ Nick-End Labeling; D051379:Mice; D018345:Mice, Knockout; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D011839:Radiation, Ionizing; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D015996:Survival Rate
D018797:Cell Cycle Proteins; D019384:Nucleic Acid Synthesis Inhibitors; C069930:rad9 protein; D006918:Hydroxyurea