Enhanced genomic instability and defective postreplication repair in RAD18 knockout mouse embryonic stem cells.
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 approximately 20-fold higher in knockout cells, and gene targeting occurred with approximately 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.
Tateishi
Satoshi
S
Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 862-0976, USA.
Niwa
Hitoshi
H
Miyazaki
Jun-Ichi
J
Fujimoto
Shiho
S
Inoue
Hirokazu
H
Yamaizumi
Masaru
M
D020816:Amino Acid Motifs; D000818:Animals; D019556:COS Cells; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008025:Ligases / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008957:Models, Genetic; D009153:Mutagens / Q000494:pharmacology; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011995:Recombination, Genetic; D029701:Saccharomyces cerevisiae Proteins; D012854:Sister Chromatid Exchange; D013234:Stem Cells / Q000166:cytology; D013997:Time Factors; D014162:Transfection; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D014466:Ultraviolet Rays; D014965:X-Rays
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D009153:Mutagens; C063607:RAD18 protein, S cerevisiae; C485954:RAD18 protein, human; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D005982:Glutathione Transferase; D008025:Ligases