I-SceI-Mediated Double-Strand Break Does Not Increase the Frequency of Homologous Recombination at the Dct Locus in Mouse Embryonic Stem Cells

Targeted induction of double-strand breaks (DSBs) at natural endogenous loci was shown to increase the rate of gene replacement by homologous recombination in mouse embryonic stem cells. The gene encoding dopachrome tautomerase (Dct) is specifically expressed in melanocytes and their precursors. To construct a genetic tool allowing the replacement of Dct gene by any gene of interest, we generated an embryonic stem cell line carrying the recognition site for the yeast I-SceI meganuclease embedded in the Dct genomic segment. The embryonic stem cell line was electroporated with an I-SceI expression plasmid, and a template for the DSB-repair process that carried sequence homologies to the Dct target. The I-SceI meganuclease was indeed able to introduce a DSB at the Dct locus in live embryonic stem cells. However, the level of gene targeting was not improved by the DSB induction, indicating a limited capacity of I-SceI to mediate homologous recombination at the Dct locus. These data suggest that homologous recombination by meganuclease-induced DSB may be locus dependent in mammalian cells

Dipoid-Specific Genome Stability Genes of S. cerevisiae: Genomic Screen Reveals Haploidization as an Escape from Persisting DNA Rearrangement Stress

Maintaining a stable genome is one of the most important tasks of every living cell and the mechanisms ensuring it are similar in all of them. The events leading to changes in DNA sequence (mutations) in diploid cells occur one to two orders of magnitude more frequently than in haploid cells. The majority of those events lead to loss of heterozygosity at the mutagenesis marker, thus diploid-specific genome stability mechanisms can be anticipated. In a new global screen for spontaneous loss of function at heterozygous forward mutagenesis marker locus, employing three different mutagenesis markers, we selected genes whose deletion causes genetic instability in diploid Saccharomyces cerevisiae cells. We have found numerous genes connected with DNA replication and repair, remodeling of chromatin, cell cycle control, stress response, and in particular the structural maintenance of chromosome complexes. We have also identified 59 uncharacterized or dubious ORFs, which show the genome instability phenotype when deleted. For one of the strongest mutators revealed in our screen, ctf18Δ/ctf18Δ the genome instability manifests as a tendency to lose the whole set of chromosomes. We postulate that this phenomenon might diminish the devastating effects of DNA rearrangements, thereby increasing the cell's chances of surviving stressful conditions. We believe that numerous new genes implicated in genome maintenance, together with newly discovered phenomenon of ploidy reduction, will help revealing novel molecular processes involved in the genome stability of diploid cells. They also provide the clues in the quest for new therapeutic targets to cure human genome instability-related diseases

The Homologous Recombination Machinery Orchestrates Post-replication DNA Repair During Self-renewal of Mouse Embryonic Stem Cells

Abstract Embryonic stem (ES) cells require homologous recombination (HR) to cope with genomic instability caused during self-renewal. Here, we report expression dynamics and localization of endogenous HR factors in DNA break repair of ES cells. In addition, we analyzed gene expression patterns of HR-related factors at the transcript level with RNA-sequencing experiments. We showed that ES cells constitutively expressed diverse HR proteins throughout the cell cycle and that HR protein expression was not significantly changed even in the DNA damaging conditions. We further analyzed that depleting Rad51 resulted in the accumulation of larger single-stranded DNA (ssDNA) gaps, but did not perturb DNA replication, indicating that ES cells were able to enter the G2-phase in the presence of unrepaired DNA gaps, consistent with the possibility that post-replication repair helps avoid stalling at the G2/M checkpoint. Interestingly, caffeine treatment inhibited the formation of Rad51 or Rad54 foci, but not the formation of γH2AX and Exo1 foci, which led to incomplete HR in ssDNA, thus increasing DNA damage sensitivity. Our results suggested that ES cells possess conserved HR-promoting machinery to ensure effective recruitment of the HR proteins to DNA breaks, thereby driving proper chromosome duplication and cell cycle progression in ES cells