A mRad51-GFP antimorphic allele affects homologous recombination and DNA damage sensitivity.

DNA Repair (Amst)

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:

Published: January 2015

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.

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Source
http://dx.doi.org/10.1016/j.dnarep.2014.11.002DOI Listing

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