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

Abstract

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.

ACKNOWLEDGMENTS

This work was supported by a Grant-in Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, and Culture of Japan; by a research grant of the Princess Takamatsu Cancer Research Fund; and by a grant from the Sagawa Foundation for Promotion of Frontier Science, Kumamoto, Japan.

We thank Kimi Araki and Ken-ichi Yamamura for providing the targeting vector pU16, pPGKpuro and pCAGGS-CRE. We thank Hideyuki Ogawa and Shunichi Takeda for valuable discussions, David Catcheside and Nell Kennedy for critical reading of the manuscript, and Chie Tateishi and Miyoko Ishizaka for technical assistance.