Elucidating the mechanisms of DNA single strand break (SSB) repair in plants

• Applicant: Professor Dr. Holger Puchta
• Subject Area: Plant Genetics and Genomics ; Plant Physiology
• Term: from 2017 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 372598077

Single strand breaks (SSBs) are a much more common DNA lesions than double strand breaks (DSBs) in the genomes of all organism. Due to the availability of highly specific endonucleases, DSB repair has been analyzed in great detail over the last 25 years in multicellular eukaryotes. This applies for the repair mechanisms as well as for the proteins involved. Only recently it became possible by the use of the CRISPR/Cas system to induce highly efficiently unique SSBs at any genomic site. Using this technology the current proposals aims to elucidate the nature of SSB repair in the model plant Arabidopsis thaliana. We were already able to show that that a single SSBs drastically induces homologous recombination (HR) between repeated sequences. In the current project we will now analyze by application of available mutants which repair and recombination proteins are involved in this SSB induced repair pathway in plants. As we could show previously that T DNA can integrate into preformed DSBs, we want to test whether also SSBs can initiate T DNA integration site specifically. Moreover, in contrast to a unique SSB, we found out that two SSBs in closer proximity (up to 100 nts) to each other in the two opposite DNA stands are highly mutagenic, producing deletions and by fill in synthesis insertions, too. We are now aiming to define the plant factors responsible for the formation of the different mutant classes. Another important question is how far two adjacent SSBs have to be apart that they are no longer recognized by the cell as coupled SSBs inducing mutagenic DNA repair. For that we will induce SSBs at various distances from 100 bps to a 2 kb (or even longer if required) that result in either 3 or 5 overhangs. We will test eu and heterochromatic regions. Besides elucidating the basic mechanism of SSB repair in plants the results of this study will also be highly relevant for the development of Cas9 nickase based genome engineering in plants.

• DFG Programme: Research Grants