Study : IDN2 Interacts with RPA and Facilitates DNA Double-Strand Break Repair by Homologous Recombination in Arabidopsis


IDN2 Interacts with RPA and Facilitates DNA Double-Strand Break Repair by Homologous Recombination in Arabidopsis
Repair of DNA double-strand break (DSB) is critical for the maintenance of genome integrity. We have previously shown that a class of DSB-induced small RNAs (diRNAs) facilitates homologous recombination (HR)-mediated DSB repair in Arabidopsis thaliana. Here we show that INVOLVED IN DE NOVO 2 (IDN2), a double-stranded RNA (dsRNA) binding protein involved in small RNA-directed DNA methylation, is required for DSB repair in Arabidopsis. We find that IDN2 interacts with the heterotrimeric replication protein A (RPA) complex. Depletion of IDN2 or the diRNA-binding ARGONAUTE 2 (AGO2) leads to increased accumulation of RPA at DSB sites and mislocalization of the recombination factor RAD51. These findings support a model in which IDN2 interacts with RPA and facilitates the release of RPA from ssDNA tails and subsequent recruitment of RAD51 at DSB sites to promote DSB repair. Overall design: Here we examined the DSB repair rate via introducing a well-established DGU.US reporter system in idn2-3, mutant background. This system consists of the DGU.US reporter (R) line containing a unique I-SceI recognition site within the direct repeats and the DSB-triggering (T) line that expresses the endonuclease I-SceI. In the crossed (RxT) line, DSBs are generated at the I-SceI site and the repair of DSBs results in restored expression of β-glucuronidase (GUS). To compare the DSB repair efficiency in a mutant background with that in the corresponding wild-type background, we first crossed both the R line and T line with idn2-3 mutant independently. After F2 segregation, the following progenies were identified by genotyping: those homozygous for either the recombination substrate locus from the R line or the I-SceI expressing locus from the T line in the respective homozygous mutant background (named R/R; idn2-3 (-/-) and T/T; idn2-3 (-/-), respectively) and those in the WT background (named R/R; IDN2 (+/+) and T/T; IDN2 (+/+), respectively). Then, plants R/R; idn2-3 (-/-) were crossed with T/T; idn2-3 (-/-) to obtain RxT; idn2-3 (-/-), whereas the corresponding WT siblings R/R; IDN2 (+/+) were crossed with T/T; IDN2 (+/+) to obtain RxT; IDN2 (+/+). The seeds of these crosses were harvested, sterilized, and sown on MS medium. Thirteen-day-old seedlings were collected for RNA extraction. To evaluate the effects of IDN2 mutation on diRNA production, small RNAs from RxT; IDN2 (+/+) and RxT; idn2-3 (-/-) plants were cloned and sequenced on Illumina Genome Analyzer IIx platform.
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