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Molecular and Cellular Biology, August 2004, p. 7235-7248, Vol. 24, No. 16
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.16.7235-7248.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Deletion of Mouse Rad9 Causes Abnormal Cellular Responses to DNA Damage, Genomic Instability, and Embryonic Lethality

Kevin M. Hopkins,¹ Wojtek Auerbach,^2, Xiang Yuan Wang,³ M. Prakash Hande,^1, Haiying Hang,¹ Debra J. Wolgemuth,³ Alexandra L. Joyner,² and Howard B. Lieberman^1*

Center for Radiological Research, College of Physicians and Surgeons,¹ Departments of Genetics & Development and Obstetrics & Gynecology and The Institute for Human Nutrition Columbia University, New York, New York 10032,³ Developmental Genetics Program, Skirball Institute of Biomedical Medicine and Howard Hughes Medical Institute, New York University School of Medicine, New York 10016²

Received 29 April 2003/ Returned for modification 12 June 2003/ Accepted 14 May 2004

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 G₂ 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.

Present address: Regeneron Pharmaceuticals, Inc., Tarrytown, NY 10591.

Present address: Department of Physiology, Faculty of Medicine, National University of Singapore, Singapore 117597, Singapore.

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