Samenvatting

Recent studies showed that in teb mutants (teb2 & teb5 (mutant allele 2 and 5 of the Teb gene)) no T-DNA integration was detectable, meaning that if there still is any T-DNA integration it will be in very low frequencies (Kregten et al., 2016). This suggests that the Teb gene is essentially involved in the integration of T-DNA.
As graduation project I will first perform a phenotypic analysis after DNA damage of Bleomycin and Methyl Methanesulphonate (MMS) of teb5, ku80 and double mutant teb5ku80 compared to the wild type (WT). The teb5 mutant has a disrupted a-NHEJ DNA repair pathway, leaving the c-NHEJ and HR pathway as the only two repair pathways. The ku80 mutant has a disrupted c-NHEJ DNA repair pathway, leaving the a-NHEJ and HR pathway as the only two repair pathways. By creating a double mutant via crossbreeding teb5 and ku80 both a-NHEJ as c-NHEJ are inactivated.
Subequently I will determine if repair of DSBs induced by artificial nucleases in teb5 mutants could be repaired by performing ‘Foot print Analyses’. These results will give an indication whether the same repair pathway is used for T-DNA integration and repair of artificially induced DSBs.
Few studies indicate that in pol-θ mutants HR still occurs, in some cases even more then in WT (Mateos-Gomez et al., 2015), (Ceccaldi et al., 2015). Also as a part of my graduation project I will determine if this is also true in plants by studying intrachromosomal recombination via the GUS assay (Gherbi et al., 2001) and integration of T-DNA via HR in the Arabidopsis thaliana Root Transformation experiments (Vergunst, Lier, Dulk-ras, & Hooykaas, 2003).