Elucidating the mechanisms of DNA single strand break (SSB) repair in plants
Plant Physiology
Final Report Abstract
Single strand breaks (SSBs) in nuclear DNA occur naturally at orders of magnitude higher frequency than double strands breaks (DSBs). DSB repair has been studied in great detail over the last 30 years whereas very little information was obtained about SSB repair and its influence on genome stability. The development of the CRISPR/Cas9 nickase enabled us now to analyse different aspect of SSB repair in plants. We were able before to link T-DNA integration to DSB repair by showing that T-DNA can be detected at sites in the plant genome were DSB have been induce during Agrobacterium transformation. We now tested whether SSB can also be used as initiation sites by using digital PCR to compare T-DNA integration into DSB and SSB at the same site into the tobacco genome. We were able to detect DSB induced T-DNA integration but no SSB induced events. Thus, the frequency of T-DNA integration in SSBs is below the experimental limit of detection and thus occurs less frequently than DSB-mediated T-DNA capture. Before, we could also show that SSBs induced by the CRISPR/Cas9 nickase at a distance of 50 to 100 bps on opposite strands are highly mutagenic leading to InDels (insertions/deletions), with insertions mainly occurring as direct tandem duplications. As short tandem repeats are overrepresented in plant genomes, this mechanism seems to have an important influence on genome evolution. We investigated up to which distance staggered SSBs are mutagenic and which DNA repair factors are essential for insertion formation in Arabidopsis. We were able to detect InDel formation up to a distance of 250 bp, although with reduced efficiency. Surprisingly, the loss of the classical non-homologous end joining (NHEJ) pathway factors KU70 or LIG4 completely abolished tandem repeat formation. The microhomology-mediated NHEJ factor POLQ was required only for patch-like insertions, which are well-known from DSB repair. As SSBs can also be repaired by the use of homology, we furthermore asked whether the classical homologous recombination (HR) pathway is involved in this process in plants. The fact that AtRAD54 is not required for homologymediated SSB repair demonstrates that also the mechanism for DSB and SSB induced HR differ drastically in plants.
