Homologous recombination (HR) is an essential biological process, which plays a pivotal role in mechanisms such as meiotic crossover and the reparation of DNA double strand breaks. In addition to being a fundamental process of life, it is also the basis of gene targeting, a technical application, which utilizes the cells HR apparatus for precise genome editing. While GT has been successfully employed for functional genome analysis in mice and yeast, higher plants are innately inefficient in GT. This is because they repair DNA double strand breaks preferentially with another pathway, non homologous end joining (NHEJ), which does not lead to targeted insertions. On the other hand, the moss Physcomitrella patens preferentially uses HR for DSB repair as well as transgene integration. This feature makes P. patens a suitable model organism for studying HR in general, which could also provide a better understanding of its outstanding GT properties.
In order to extend our knowledge on this subject, the present thesis focused on two major topics. In the first part the inventory of DNA repair and recombination genes in P. patens was assessed via a BLAST based approach, which was made possible by the recent release of the P. patens genome draft. Intriguingly, P. patens possesses all important NHEJ genes, therefore its bias for HR over NHEJ could not be explained based on simple presence or absence of known NHEJ genes. On the other hand, there were similarities regarding the absence/presence of HR genes between P. patens and the also highly HR biased and GT efficient yeast. For example, BRCA1, BRCA2 and BARD1 are neither found in P. patens nor in yeast. Even more intriguingly they are available in Arabidopsis and also humans, where NHEJ is the dominant pathway. A further remarkable observation was that for 33 of the DNA repair and recombination related genes available in P. patens additional paralogs were identified. These paralogs are certainly interesting candidates for further studies.
The second part of the thesis focused on the ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) kinases, two key upstream signaling components of HR mediated DNA DSB repair. Utilizing the proficiency of P. patens in gene targeting ATM and ATR single as well as double mutants were generated by disrupting their kinase domains. These mutants were then used to characterize the function of the P. patens ATM and ATR homologs. Observing the mutant lines it was found that the loss of ATR function severely compromised vegetative and reproductive development while ATM disruption had just a mild effect on vegetative development. Testing the sensitivity of the mutant lines to various DNA DSB inducing agents it was found that in P. patens mainly ATR is required to facilitate the repair of these lesions, a situation similar to yeast but different to human cells and Arabidopsis, where also loss of ATM markedly affects sensitivity to these genotoxic agents. Similarly, another response to DNA DSB, the arrest of the cell cycle is also mediated by largely ATR in P. patens as well as yeast, while in Arabidopsis and human cells ATM also plays an important role. As part of the mutant phenotype analysis, the effect of ATM and ATR loss of function on the mode of transgene integration was tested. It was found that removal of ATM function significantly increased NHEJ mediated random integrations while targeted integrations were unaffected, the latter were reduced only in the ATM and ATR double mutant.
Another major part of the thesis was to assess the global transcriptional change in response to bleomycin mediated DNA DSB induction in wild type as well as in mutant lines. Although P. patens is known to be biased towards HR, unexpectedly, in addition to HR genes also NHEJ genes were found transcriptionally induced in the wild type. Comparing the transcriptomes of wild type and mutant lines large differences in transcript levels were already observed without bleomycin treatment. Additional bleomycin treatment showed that a large number of DNA damage responsive transcripts could not be induced or repressed in the mutant lines. Another interesting finding was that while the transcriptional DNA damage response in Arabidopsis appears to largely rely on ATM function, in P. patens ATR seem to be more important in this regard
