title: A mRad51-GFP antimorphic allele affects homologous recombination and DNA damage sensitivity.
abstract: Accurate DNA double-strand break repair through homologous recombination is essential for preserving genome integrity. Disruption of the gene encoding RAD51, the protein that catalyzes DNA strand exchange during homologous recombination, results in lethality of mammalian cells. Proteins required for homologous recombination, also play an important role during DNA replication. To explore the role of RAD51 in DNA replication and DSB repair, we used a knock-in strategy to express a carboxy-terminal fusion of green fluorescent protein to mouse RAD51 (mRAD51-GFP) in mouse embryonic stem cells. Compared to wild-type cells, heterozygous mRad51(+/wt-GFP) embryonic stem cells showed increased sensitivity to DNA damage induced by ionizing radiation and mitomycin C. Moreover, gene targeting was found to be severely impaired in mRad51(+/wt-GFP) embryonic stem cells. Furthermore, we found that mRAD51-GFP foci were not stably associated with chromatin. From these experiments we conclude that this mRad51-GFP allele is an antimorphic allele. When this allele is present in a heterozygous condition over wild-type mRad51, embryonic stem cells are proficient in DNA replication but display defects in homologous recombination and DNA damage repair.
authors.affiliation: Department of Reproduction and Development, Erasmus MC, PO Box 2040, 3000 CA Rotterdam, The Netherlands.
authors.forename: Evert-Jan
authors.initials: EJ
authors.lastname: Uringa
authors.affiliation: Department of Genetics, Cancer Genomics Center, Erasmus MC, PO Box 2040, 3000 CA Rotterdam, The Netherlands.
authors.forename: Céline
authors.initials: C
authors.lastname: Baldeyron
authors.affiliation: Department of Genetics, Cancer Genomics Center, Erasmus MC, PO Box 2040, 3000 CA Rotterdam, The Netherlands.
authors.forename: Hanny
authors.initials: H
authors.lastname: Odijk
authors.affiliation: Department of Reproduction and Development, Erasmus MC, PO Box 2040, 3000 CA Rotterdam, The Netherlands.
authors.forename: Evelyne
authors.initials: E
authors.lastname: Wassenaar
authors.affiliation: Erasmus Optical Imaging Centre, Department of Pathology, Erasmus MC, PO Box 2040, 3000 CA Rotterdam, The Netherlands.
authors.forename: Wiggert A
authors.initials: WA
authors.lastname: van Cappellen
authors.affiliation: Department of Genetics, Cancer Genomics Center, Erasmus MC, PO Box 2040, 3000 CA Rotterdam, The Netherlands.
authors.forename: Alex
authors.initials: A
authors.lastname: Maas
authors.affiliation: Department of Genetics, Cancer Genomics Center, Erasmus MC, PO Box 2040, 3000 CA Rotterdam, The Netherlands.
authors.forename: Jan H J
authors.initials: JH
authors.lastname: Hoeijmakers
authors.affiliation: Department of Reproduction and Development, Erasmus MC, PO Box 2040, 3000 CA Rotterdam, The Netherlands.
authors.forename: Willy M
authors.initials: WM
authors.lastname: Baarends
authors.affiliation: Department of Genetics, Cancer Genomics Center, Erasmus MC, PO Box 2040, 3000 CA Rotterdam, The Netherlands; Department of Radiation Oncology, Erasmus MC, PO Box 2040, 3000 CA Rotterdam, The Netherlands.
authors.forename: Roland
authors.initials: R
authors.lastname: Kanaar
authors.affiliation: Department of Genetics, Cancer Genomics Center, Erasmus MC, PO Box 2040, 3000 CA Rotterdam, The Netherlands; Department of Radiation Oncology, Erasmus MC, PO Box 2040, 3000 CA Rotterdam, The Netherlands; Department of Surgical Oncology, Erasmus MC, PO Box 2040, 3000 CA Rotterdam, The Netherlands. Electronic address: j.essers@erasmusmc.nl.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Essers
mesh_terms: D000483:Alleles; D000818:Animals; D002478:Cells, Cultured; D004247:DNA / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D053903:DNA Breaks, Double-Stranded; D004261:DNA Replication / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006579:Heterozygote; D051379:Mice; D016685:Mitomycin / Q000494:pharmacology; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D051135:Rad51 Recombinase / Q000235:genetics* / Q000378:metabolism; D011839:Radiation, Ionizing; D059767:Recombinational DNA Repair / Q000235:genetics*
chemical_list: D050505:Mutant Proteins; D049452:Green Fluorescent Proteins; D016685:Mitomycin; D004247:DNA; D051135:Rad51 Recombinase; C495154:Rad51 protein, mouse
keywords: DNA repair; Embryonic stem cells; Homologous recombination; RAD51; Replication