RAD51 and RAD51B Play Diverse Roles in the Repair of DNA Double Strand Breaks in Physcomitrium patens

Abstract

RAD51 is involved in finding and invading homologous DNA sequences for accurate homologous recombination (HR). Its paralogs have evolved to regulate and promote RAD51 functions. The efficient gene targeting and high HR rates are unique in plants only in the moss Physcomitrium patens (P. patens). In addition to two functionally equivalent RAD51 genes (RAD1-1 and RAD51-2), other RAD51 paralogues were also identified in P. patens. For elucidation of RAD51's involvement during DSB repair, two knockout lines were constructed, one mutated in both RAD51 genes (Pprad51-1-2) and the second with mutated RAD51B gene (Pprad51B). Both lines are equally hypersensitive to bleomycin, in contrast to their very different DSB repair efficiency. Whereas DSB repair in Pprad51-1-2 is even faster than in WT, in Pprad51B, it is slow, particularly during the second phase of repair kinetic. We interpret these results as PpRAD51-1 and -2 being true functional homologs of ancestral RAD51 involved in the homology search during HR. Absence of RAD51 redirects DSB repair to the fast NHEJ pathway and leads to a reduced 5S and 18S rDNA copy number. The exact role of the RAD51B paralog remains unclear, though it is important in damage recognition and orchestrating HR response.

Keywords: DNA double-strand break (DSB); Physcomitrella; bleomycin; comet assay; evolutionary divergence; homologous recombination (HR); non-homologous end-joining (NHEJ); rDNA; repair kinetic.

Figure 1

Simplified phylogenetic tree of RAD51 superfamily. The evolutionary history was inferred by using maximum likelihood analysis. RecA Escherichia coli was used as the outgroup. Bootstrap support values > 50% are shown on the branch above. The ML log likelihood is −16,689.642127. The analysis included 24 amino acid sequences and 673 positions in the final data set. (A) XRCC2, (B) DCM1, (C) RAD51-1 and -2, (D) RAD51D, (E) RAD51C, (F) XRCC3, and (G) RAD51B. Gray boxes depict RAD51-1 and -2 and RAD51B clads. Accession numbers are listed in Supplementary Table S1.

Figure 2

Characterization of moss Pprad51s mutant lines. (A) Morphology of 1-month-old colonies of WT and Pprad51B and Pprad51-1-2 mutant lines grown on BCDAT medium without bleomycin treatment. (B) Seven-day-old protonemata stained with propidium iodide. (C) The growth rates of WT and mutant lines measured as fresh weight of 3-week-old untreated plants. (D) Loss of 5S and 18S rDNA genomic copies in WT and mutant lines. Relative rDNA copy numbers were measured by qPCR and normalized to values obtained in the WT. Student’s t-test: * p < 0.05; ** p < 0.01. Error bars represent SD.

Figure 3

Growth response of WT, Pprad51B, and Pprad51-1-2 plants to 1 h treatment with 0.5, 1, 3, 5, 10, 30, and 50 μg bleomycin/mL. After the treatment, explants were inoculated on Petri plates with drug-free BCDAT medium and grown under standard conditions for 3 weeks. For each experimental point, the weight of treated plants collected from two replicas was normalized to the weight of untreated plants and plotted as relative fresh weight, which was set as a default to 100 for untreated plants. Error bars represent SD.

Figure 4

DSB repair kinetics determined by comet assay in dividing (1d) or differentiated (7d) tissue of WT (blue), Pprad51B (orange), and Pprad51-1-2 (red) mutant lines. Protonemata, which regenerated for 1 (A) or 7 (B) days after subculture were treated with 30 μg bleomycin/mL for 1 h. Repair kinetics was measured as % of DSBs remaining after the 0, 3, 5, 10, 20, 60, and 180 min of repair recovery. Maximum damage is normalized as 100% at t = 0 for all lines. Error bars indicate SD.

Figure 5

Analysis of DSB repair data. Plotted are the parameters of the ‘Two phase decay’ kinetics model that were determined by GraphPad program Prism v.5. The half-life of first (τ_fast) and second (τ_slow) phase of the repair curve and the plateau of remaining damage are plotted in orange, gray, and blue, respectively. The numeric values are provided in the table below the plot. Student’s t-test: * p < 0.05; ** p < 0.01.