Abstract
AtRad52 homologs are involved in DNA recombination and repair, but their precise functions in different homologous recombination (HR) pathways or in gene-targeting have not been analyzed. In order to facilitate our analyses, we generated an AtRad52-1A variant that had a stronger nuclear localization than the native gene thanks to the removal of the transit peptide for mitochondrial localization and to the addition of a nuclear localization signal. Over-expression of this variant increased HR in the nucleus, compared with the native AtRad52-1A: it increased intra-chromosomal recombination and synthesis-dependent strand-annealing HR repair rates; but conversely, it repressed the single-strand annealing pathway. The effect of AtRad52-1A over-expression on gene-targeting was tested with and without the expression of small RNAs generated from an RNAi construct containing homology to the target and donor sequences. True gene-targeting events at the Arabidopsis Cruciferin locus were obtained only when combining AtRad52-1A over-expression and target/donor-specific RNAi. This suggests that sequence-specific small RNAs might be involved in AtRad52-1A-mediated HR.
| Original language | English |
|---|---|
| Pages (from-to) | 30-40 |
| Number of pages | 11 |
| Journal | Plant Journal |
| Volume | 95 |
| Issue number | 1 |
| Early online date | 18 Apr 2018 |
| DOIs | |
| Publication status | Published - Jul 2018 |
Funding
The authors thank all members of Avi Levy's laboratory in the Department of Plant Sciences at the Weizmann Institute of Science, in particular Daniela Ben‐Tov for editing the manuscript. The authors thank Dr Ron Rotkopf at the Weizmann Institute of Science for help with statistical analyses. The authors thank Prof. Holger Puchta from Karlsruhe Institute of Technology for providing recombination tester and Ubip_I‐SceI lines. This work was funded by European Union research grant (RECBREED). A.A.L. holds the Gilbert de Botton Chair of Plant Science.