Double-strand break repair in plants
Final Report Abstract
Using the rare cutting restriction endonuclease I-SceI as mean to induce double strand breaks (DSBs) in the genome a deeper understanding of the mechanisms of homologous DSB repair in plant cells was achieved. An important question for a better understanding of plant genome evolution was whether sequences that have been transcribed into RNA can also be used for the repair of a DSB at a detectable frequency. In principle, the template could be either a RNA or a cDNA produced by reverse transcriptase from the RNA. Via transcription the number of available template molecules might be drastically enhanced. To address this question experiments were performed in tobacco. An artificial kanamycin intron system was used for the assay. Tobacco plants were transformed with two different T- DNA constructs, the "Target" and the "Donor". The target contained an I-SceI site and parts of a kanamycin resistance gene and the donor an expression cassette for I-SceI under the control of a double-35S promoter and further parts of the kanamycin resistance gene. An intron was included in the donor in the part of the gene that by homologous DSB repair can be used as template to restore the functional resistance gene. In case of an RNA or cDNA being used as template the intron sequence will be removed by splicing and will not be included in the restored genomic kanamycin gene in the target locus. In case the genomic copy is used, the intron will be copied into the break, too. Performing a row of experiments we were able to find two recombination events that are due to the presence of the intron sequences of the kanamycin resistance by classical DNA recombination reactions. However, we were not able to detect any event that might have involved an RNA intermediate. This by no means proves that there is no RNA mediated DSB repair by homologous sequences at all, it only shows that it seems to occur rather infrequent - at the same or a lower rate then ectopic DNA recombination. It might therefore play no major role in genome evolution. By applying recombination substrates in which recombination is initiated by the induction of a site-specific DSB by the homing endonuclease I-SceI, we were able to characterize the involvement of different factors in synthesis-dependent strand-annealing (SDSA) and single strand annealing (SSA) pathways of HR. The nucleases MRE11 and COM1, both involved in DSB end processing, were not required for either SDSA or SSA in our assay system. Both SDSA and SSA were even more efficient without MRE11, in accordance with the fact that a loss of MRE11 might negatively affect non-homologous end joining efficiency. Loss of the classical recombinase RAD51 or its two paralogues RAD51C and XRCC3, as well as the SWI2/SNF2 remodelling factor RAD54, resulted in a drastic deficiency in SDSA but had hardly any influence on SSA, confirming that a strand exchange reaction is only required for SDSA. The SWI2/SNF2 ATPase RAD5A which is involved in postreplicative repair plays no role in SSA but is required for SDSA. The helicases RECQ4A and FANCM are surprisingly not only needed for SDSA but to a lesser extent also for SSA. The same holds true for the nuclease MUS81, indicating that the protein is to a certain extent required for the processing of intermediates in both pathways. Both SSA and SDSA were affected only weakly when the SMC6B protein, implicated in sister chromatid recombination, was absent, indicating that SSA and SDSA are in most cases intrachromatid recombination reactions.
Publications
- (2010) RAD5A, RECQ4A and MUS81 have specific functions in homologous recombination and define different pathways of DNA repair in Arabidopsis thaliana. Plant Cell 22: 3318–3330
Mannuss A., Dukowic-Schulze S., Suer S., Hartung F., Pacher M. and Puchta H.
- (2012). The requirement for recombination factors differs considerably between different pathways of homologous double-strand break repair in somatic plant cells. Plant Journal 72: 781-790
Roth N., Klimesch J., Dukowic-Schulze S., Pacher M., Mannuss A. and Puchta H.
(See online at https://doi.org/10.1111/j.1365-313X.2012.05119.x)
