Homology-dependent repair is involved in 45S rDNA loss in plant CAF-1 mutants

Abstract

Arabidopsis thaliana mutants in FAS1 and FAS2 subunits of chromatin assembly factor 1 (CAF1) show progressive loss of 45S rDNA copies and telomeres. We hypothesized that homology-dependent DNA damage repair (HDR) may contribute to the loss of these repeats in fas mutants. To test this, we generated double mutants by crossing fas mutants with knock-out mutants in RAD51B, one of the Rad51 paralogs of A. thaliana. Our results show that the absence of RAD51B decreases the rate of rDNA loss, confirming the implication of RAD51B-dependent recombination in rDNA loss in the CAF1 mutants. Interestingly, this effect is not observed for telomeric repeat loss, which thus differs from that acting in rDNA loss. Involvement of DNA damage repair in rDNA dynamics in fas mutants is further supported by accumulation of double-stranded breaks (measured as γ-H2AX foci) in 45S rDNA. Occurrence of the foci is not specific for S-phase, and is ATM-independent. While the foci in fas mutants occur both in the transcribed (intranucleolar) and non-transcribed (nucleoplasmic) fraction of rDNA, double fas rad51b mutants show a specific increase in the number of the intranucleolar foci. These results suggest that the repair of double-stranded breaks present in the transcribed rDNA region is RAD51B dependent and that this contributes to rDNA repeat loss in fas mutants, presumably via the single-stranded annealing recombination pathway. Our results also highlight the importance of proper chromatin assembly in the maintenance of genome stability.

Keywords: 45S rDNA; Arabidopsis thaliana; DNA repair; FAS1; FAS2; RAD51B; chromatin assembly factor 1; genome instability.

Figure 1

The loss of 45S rDNA and telomeres is mitotic. Telomere lengths (a) and copy numbers of 45S rDNA (b, c) were measured in consecutive passages (P1–P9) of callus cultures derived from the second generation of fas1 or fas2 mutant plants as indicated. Data obtained in G2 and G5 generations of mutant plants are shown for comparison. Relative content of 45S rDNA is expressed in arbitrary units (a.u.) with respect to the level in WT plants with no mutational history.

Figure 2

Loss of 45S rDNA repeats in fas1 (a) and fas2 (b) mutants with either functional or dysfunctional RAD51B gene. 45S rDNA copy numbers were measured by qPCR and normalized to the corresponding values obtained in WT plants. Statistical analysis and plots were performed by R software (http://www.r-project.org). The two-sided Mann–Whitney–Wilcoxon test (α = 0.05), non-paired, was used. One asterisk denotes a P-value <0.05, two asterisks denote a P-value <0.01. Thick lanes inside the box plots indicate median values.

Figure 3

Loss of telomeric DNA repeats in fas1 (a, b) and fas2 (c, d) mutants with either functional or dysfunctional RAD51B gene. Telomere lengths were measured by TRF analysis in F2, F3 and F5 generations of plants segregated from crossing between fas1/2 and rad51b mutants, and mean telomere lengths with corresponding error bars were plotted to the diagrams depicted in panels (a) and (c). Corresponding generations of parental mutant plants were analysed in parallel together with Col0 WT plants without a mutation history. Source TRF patterns of the last generation are shown in panels (b) and (d), data from the earlier generations are shown in Figure S3.

Figure 4

Sensitivity of seedlings to MMS. Loss of RAD51B neither shows sensitivity to MMS, nor increases sensitivity of fas mutants to MMS [panels (a) and (b), respectively]. 18S rDNA copy numbers and transcript levels are shown in panels (c) and (d), respectively.

Figure 5

γ-H2AX foci in fas and fas rad51b mutant plants. (a) Immunofluorescence of root tip interphase nuclei indicates γ-H2AX foci formation in fas1 and fas1 rad51b mutants. DNA is stained with DAPI (blue), γ-H2AX foci are colored in green and merged images overlay γ-H2AX foci onto chromosomes. Scale bar, 2 μm. An example of a γ-H2AX focus located outside the nucleolus is indicated by a white arrow in the fas1 mutant and an example of a focus located inside the nucleolus is indicated by a yellow arrow in the fas1 rad51b mutant. (b) Graphical representation of the number of intra- and extra-nucleolar γ-H2AX foci counted in WT, rad51b, fas1, fas1 rad51b, fas2 and fas2 rad51b plants of third and fifth mutant generations in plants with low or high number of rDNA repeats (indicated above the graph). Mean values are from counting foci of 100 interphase nuclei. Error bars indicate standard error.

Figure 6

γ-H2AX foci colocalise with 45S rDNA FISH in the fas1 rad51b mutant. (a) Immunostaining and 45S rDNA FISH labelling of root tip nuclei of fas1 and fas1 rad51b mutants. Nuclei were stained with DAPI (blue), γ-H2AX foci are colored in green and FISH signals are colored in red. Images are a single focal plane from a deconvolved three-dimensional image. Bar, 2 μm. (b) Numerical results recapitulating the number and the percentage of co-localization of γ-H2AX foci with the rDNA probe.

Figure 7

γ-H2AX foci formation does not depend on replication. (a) Immunofluorescence of root tip nuclei labelled with EdU showing γ-H2AX foci formation in WT, fas1 and fas1 rad51b replicated nuclei. DNA is stained with DAPI (blue), EdU incorporation in red and γ-H2AX foci in green. Scale bar, 2 μm. (b) Graphical representation of results for the appearance of γ-H2AX foci in total interphase nuclei or in replicating (EdU+) nuclei of fas1 or fas1 rad51b mutant. Mean numbers of foci counted on 100 interphase or S-phase nuclei. Error bars indicate standard error.

Figure 8

γ-H2AX foci in fas1 and fas1rad51b mutants are ATR dependent. Graphical representation of the number of γ-H2AX counted inside or outside the nucleolus in WT, rad51b, fas1, fas1 rad51b, fas2 and fas2 rad51b, with or without IATM. Mean numbers of foci counted on 100 interphase nuclei. Error bars indicate standard error.