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Molecular and Cellular Biology, January 2003, p. 474-481, Vol. 23, No. 2
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.2.474-481.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Enhanced Genomic Instability and Defective Postreplication Repair in RAD18 Knockout Mouse Embryonic Stem Cells

Satoshi Tateishi,1 Hitoshi Niwa,2 Jun-Ichi Miyazaki,3 Shiho Fujimoto,1 Hirokazu Inoue,4 and Masaru Yamaizumi1*

Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 862-0976,1 Laboratory of Pluripotent Cell Studies, RIKEN Center for Developmental Biology, Kobe 650-0047,2 Department of Nutrition and Physiological Chemistry, Osaka University School of Medicine, Osaka 565-0871,3 Department of Regulation Biology, Faculty of Science, Saitama University, Urawa 338-8570, Japan4

Received 14 May 2002/ Returned for modification 23 September 2002/ Accepted 21 October 2002

In lower eukaryotes, Rad18 plays a crucial role in postreplication repair. Previously, we isolated a human homologue of RAD18 (hRAD18) and showed that human cells overexpressing hRad18 protein with a mutation in the RING finger motif are defective in postreplication repair. Here, we report the construction of RAD18-knockout mouse embryonic stem cells by gene targeting. These cells had almost the same growth rate as wild-type cells and manifested phenotypes similar to those of human cells expressing mutant Rad18 protein: hypersensitivity to multiple DNA damaging agents and a defect in postreplication repair. Mutation was not induced in the knockout cells with any higher frequencies than in wild-type cells, as shown by ouabain resistance. In the knockout cells, spontaneous sister chromatid exchange (SCE) occurred with twice the frequency observed in normal cells. After mild DNA damage, SCE was threefold higher in the knockout cells, while no increase was observed in normal cells. Stable transformation efficiencies were ~20-fold higher in knockout cells, and gene targeting occurred with ~40-fold-higher frequency than in wild-type cells at the Oct3/4 locus. These results indicate that dysfunction of Rad18 greatly increases both the frequency of homologous as well as illegitimate recombination, and that RAD18 contributes to maintenance of genomic stability through postreplication repair.

* Corresponding author. Mailing address: Institute of Molecular Embryology and Genetics, Kumamoto University, Kuhonji 4-24-1, Kumamoto 862-0976, Japan. Phone: (81) 96 373 6601. Fax: (81) 96 373 6604. E-mail: yamaizm{at}gpo.kumamoto-u.ac.jp.

Molecular and Cellular Biology, January 2003, p. 474-481, Vol. 23, No. 2
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.2.474-481.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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