The plant-specific CDKB1-CYCB1 complex mediates homologous recombination repair in Arabidopsis

Abstract

Upon DNA damage, cyclin-dependent kinases (CDKs) are typically inhibited to block cell division. In many organisms, however, it has been found that CDK activity is required for DNA repair, especially for homology-dependent repair (HR), resulting in the conundrum how mitotic arrest and repair can be reconciled. Here, we show that Arabidopsis thaliana solves this dilemma by a division of labor strategy. We identify the plant-specific B1-type CDKs (CDKB1s) and the class of B1-type cyclins (CYCB1s) as major regulators of HR in plants. We find that RADIATION SENSITIVE 51 (RAD51), a core mediator of HR, is a substrate of CDKB1-CYCB1 complexes. Conversely, mutants in CDKB1 and CYCB1 fail to recruit RAD51 to damaged DNA CYCB1;1 is specifically activated after DNA damage and we show that this activation is directly controlled by SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1), a transcription factor that acts similarly to p53 in animals. Thus, while the major mitotic cell-cycle activity is blocked after DNA damage, CDKB1-CYCB1 complexes are specifically activated to mediate HR.

Keywords: CDK; DNA damage; cell cycle; cyclin; homologous recombination.

Figure EV1. T‐DNA insertion mutants of the Arabidopsis thaliana CYCB1 genes

1. Schematic overview of T‐DNA insertions for cycb1;1, cycb1;2, cycb1;3, and cycb1;4. Black boxes display exons, and red arrowheads indicate the positions of the T‐DNA insertion.

2. Quantitative PCR for relative expression levels in the wild type (Col‐0), cycb1;1, cycb1;2 and cycb1;4. EXP, SAND, and ACT7 were used as reference genes for normalization. Each value represents the mean ± standard deviation of three independent experiments.

3. Quantitative PCR for relative expression levels in the wild type (Nos‐0) and cycb1;3. EXP, SAND, and ACT7 were used as reference genes for normalization. Each value represents the mean ± standard deviation of three independent experiments.

Figure 1. Mutants of B1‐type cyclins are hypersensitive to cisplatin

1. A

   cycb1;1, cycb1;2, cycb1;3, cycb1;4, cycb1;1/1;2, cycb1;1/1;3, cycb1;1/1;4, cycb1;2/1;4, cycb1;3/1;4, and the wild type (from left to right) on control plates without genotoxic agent 10 days after germination.

2. B

   The wild type, single, and double mutants of cycb1 were grown on control plates without genotoxic agent. Root lengths were measured 10 days after germination.

3. C

   The wild type, cycb1;1, cycb1;2, cycb1;3, cycb1;4, cycb1;1/1;2, cycb1;1/1;3, cycb1;1/1;4, cycb1;2/1;4, cycb1;3/1;4 (from left to right) on plates containing 1 mM hydroxyurea (HU) 10 days after germination. The rightmost plant is the wee1 mutant that shows high sensitivity to HU.

4. D

   The wild type, single, and double mutants of cycb1 were grown on plates supplemented with 1 mM HU. Root lengths were measured 10 days after germination.

5. E

   The wild type, cycb1;1, cycb1;2, cycb1;3, cycb1;4, cycb1;1/1;2, cycb1;1/1;3, cycb1;1/1;4, cycb1;2/1;4, cycb1;3/1;4 (from left to right) on plates containing 0.6 μg/ml bleomycin (BLM) 10 days after germination. The rightmost plant is the ku70 mutant that shows high sensitivity to BLM.

6. F

   The wild type, single, and double mutants of cycb1 were grown on plates supplemented with 0.6 μg/ml BLM. Root lengths were measured 10 days after germination.

7. G

   The wild type, cycb1;1, cycb1;2, cycb1;3, cycb1;4, cycb1;1/1;2, cycb1;1/1;3, cycb1;1/1;4, cycb1;2/1;4, cycb1;3/1;4 (from left to right) on plates containing 15 μM cisplatin 6 days after germination, that is, 3 days after transfer from control plates.

8. H–J

   cycb1 mutants were germinated on control plates and were transferred to new control plates (H) or plates supplemented with 15 μM (I) or 30 μM (J) cisplatin 3 days after germination. Root lengths were measured 3 days after transfer and the net root growth of 3 days is shown in the graphs.

9. K

   The wild type, cycb1;1, cycb1;2, cycb1;3, cycb1;4, cycb1;1/1;2, cycb1;1/1;3, cycb1;1/1;4, cycb1;2/1;4, cycb1;3/1;4 (from left to right) on plates containing 30 μM cisplatin 6 days after germination, that is, 3 days after transfer from control plates.

10. L–N

    cycb1 double mutants germinated on control plates and were transferred to new control plates (L) or plates supplemented with 15 μM (M) or 30 μM (N) cisplatin 3 days after germination. Root lengths were measured 3 days after transfer and the net root growth of 3 days is shown in the graphs.

Data information: One or two asterisks indicate significant differences within a 5 and 1% confidence interval, respectively (Student's t‐test). Scale bars: 1 cm. Three biological replicates, each containing at least 15 plants, were analyzed. The mean of the root length of each individual experiment was determined and again averaged for the three biological replicates. Graphs represent mean ± SD.

Figure EV2. Phenotype of cycb1 mutants under greenhouse conditions

1. A–D

   Single mutants cycb1;1 (A), cycb1;2 (B), cycb1;3 (C), and cycb1;4 (D).

2. E–G

   Double mutants cycb1;1 cycb1;2 (E), cycb1;1 ku70 (F), and cycb1;2 ku70 (G). Seedlings of cycb1;1 cycb1;2 are smaller and more diverse than wild‐type plants.

3. H

   The triple mutant cycb1;1 cycb1;2 ku70 displays a similar phenotype as the double mutant cycb1;1 cycb1;2.

4. I

   Wild type.

5. J

   Single mutant ku70.

Data information: All images were taken 14 days after germination. Scale bars: 1 cm.

Figure EV3. Root growth ratios of cycb1 and cdkb1 mutants

1. A, B

   Graphs represent the ratio of the mean growth rate on 1 mM hydroxyurea (HU) and 0.6 μg/ml bleomycin (BLM) compared to control experiments on plates lacking genotoxins for the wild type and the double mutant cycb1;1 cycb1;2 (A) or the weak CDKA;1 allele DE (CDKA;1 ^T15D;Y15E) (B).

2. C

   Graphs represent the ratio of the mean growth rate on 1 mM HU and 0.6 μg/ml BLM compared to control experiments on plates lacking genotoxins for the wild type, the single mutants cdkb1;1 and cdkb1;2, and the double mutant cdkb1;1 cdkb1;2.

Figure EV4. Cell death in cycb1 mutants after cisplatin treatment

1. A–L

   Propidium iodide staining of the apical root meristem in wild type grown on control plates (A) or plates supplemented with 50 μM cisplatin for 24 h (B); cycb1;1 grown on control plates (C) or plates supplemented with 50 μM cisplatin for 24 h (D); cycb1;2 grown on control plates (E) or plates supplemented with 50 μM cisplatin for 24 h (F); cycb1;3 grown on control plates (G) or plates supplemented with 50 μM cisplatin for 24 h (H); cycb1;4 grown on control plates (I) or plates supplemented with 50 μM cisplatin for 24 h (J); cycb1;1 cycb1;2 grown on control plates (K) or plates supplemented with 50 μM cisplatin for 24 h (L). Red spots show cell death. Scale bars: 20 μm.

Figure EV5. Ploidy levels in the wild type, cdkb1, and cycb1 mutants

Flow cytometry analysis of root tips of the wild type and the double mutants cdkb1;1 cdkb1;2 and cycb1;1 cycb1;3 under control conditions without genotoxins and transferred 5 days after germination to plates supplemented with 50 μM cisplatin for 24 h.

Figure EV6. The triple mutant cyca2;2 cyca2;3 cyca2;4 is not hypersensitive to hydroxyurea, bleomycin, and cisplatin

1. The wild type and the triple mutant cyca2;234 were grown on control plates or plates containing 1 mM hydroxyurea (HU) or 0.6 μg/ml bleomycin (BLM) for 10 days. Root lengths were measured 10 days after germination. The mutants wee1 and ku70 were used as positive controls for hydroxyurea and bleomycin sensitivity, respectively.

2. The wild type and cyca2;234 triple mutant germinated on control plates without cisplatin and were transferred to plates containing 30 μM cisplatin 3 days after germination. Roots were measured 3 days after transfer and the net growth of 3 days is shown in the graph.

3. Images show the wild type and cyca2;234 triple mutant on plates supplemented with 30 μM cisplatin. Images were taken 6 days after germination, that is, 3 days after transfer to cisplatin. Scale bar: 0.5 cm.

Data information: One or two asterisks indicate significant differences within a 5% and 1% confidence interval, respectively (Student's t‐test). Three biological replicates, each containing at least 15 plants, were analyzed. The mean of the root length of each individual experiment was determined and again averaged for the three biological replicates. Graphs represent mean ± SD.

Figure 2. Mutants of cdkb1 and cycb1 show increased number of DSBs and delayed DNA repair upon cisplatin treatment

1. Representative examples of comets of 21‐day‐old wild‐type plants, cdkb1;1 cdkb1;2 and cycb1;1 cycb1;3 double mutant seedlings in full spectrum view of the TriTek Comet Score software. Shown are comets of plants incubated with 50 μM cisplatin for 1 h and then transferred to medium without cisplatin for 30 min (recovery) and plants incubated without cisplatin for 1 h (control), respectively.

2. Box plot of percentage of tail DNA of wild‐type cells, cdkb1;1 cdkb1;2 and cycb1;1 cycb1;3 double mutants under cisplatin treatment. Plots are based on analyses of 200 cells per sample from random microscopic fields of three independent biological replicates. The percentage of DNA fragments in the comet tail was calculated by the TriTek Comet Score software. The box represents the interquartile range, the line across the box indicates the median values, and whiskers represent 5–95 percentile values. Brackets connect plots of sample groups that are significantly different with a confidence level higher than 99.99% calculated with Student's t‐test.

3. Immunostaining of γ‐H2AX foci in wild‐type plants and mutant cells after 2 h of treatment with 50 μM cisplatin.

4. Counted numbers of γ‐H2AX foci per cell detected after 2 h of treatment with 50 μM cisplatin in wild‐type and mutant plants. For each sample, the γ‐H2AX foci of 100 cells were counted and grouped into six categories: cells with no, 1–2, 3–5, 6–10, 11–20, and more than 20 foci per cell.

Figure 3. Homologous recombination frequencies are strongly reduced in cycb1 mutants

1. A

   Schematic drawing of homologous recombination assay. Restoring the functional GUS gene from two disrupted parts (GU' and US') is restricted to an intermolecular homologous recombination event. Homologous events occur only when a sister chromatid or homolog is available as a template, that is, in G2 phase of the cell cycle.

2. B

   Wild‐type plants show blue spots on the leaves after 3 days of incubation on 30 μM cisplatin. Arrows indicate representative blue sectors.

3. C

   cycb1;1 plants show blue spots on the leaves after 3 days of incubation on 30 μM cisplatin. Arrows indicate representative blue sectors.

4. D–F

   Graphs show numbers of blue sectors per plant grown without drug treatment (D) or after incubation on 15 μM (E) or 30 μM (F) cisplatin for 3 days. One or two asterisks indicate significant differences within a 5 and 1% confidence interval, respectively (Student's t‐test). Three biological replicates, each containing at least 15 plants, were analyzed. The mean of the root length of each individual experiment was determined and again averaged for the three biological replicates. Graphs represent mean ± SD.

Figure 4. Mutants of cdkb1 but not cdka;1 are hypersensitive to cisplatin

1. A–C

   The wild type and CDKA;1‐DE mutants were grown on control plates (A) or containing 1 mM hydroxyurea (B) or 0.6 μg/ml bleomycin (C) for 10 days. The mutants wee1 and ku70 were used as positive controls for hydroxyurea or bleomycin sensitivity, respectively. Root lengths were measured 10 days after germination.

2. D–F

   The wild type, cdkb1;1, cdkb1;2, and the double mutant cdkb1;1 cdkb1;2 were grown on control plates (D) or plates containing 1 mM hydroxyurea (E) or 0.6 μg/ml bleomycin (F) for 10 days. The mutants wee1 and ku70 were used as positive controls for hydroxyurea and bleomycin sensitivity, respectively. Root lengths were measured 10 days after germination.

3. G

   The wild type, CDKA;1‐DE, and the double mutant cdkb1;1 cdkb1;2 were grown on control plates and were transferred to plates containing 15 or 30 μM cisplatin 3 days after germination. Root lengths were measured 3 days after transfer and the net root growth of 3 days is shown in the graphs.

4. H, I

   Graphs represent the ratio of the mean growth rate on 15 μM (H) or 30 μM (I) cisplatin compared to control experiments on plates lacking cisplatin for the wild type, CDKA;1‐DE, and cdkb1;1 cdkb1;2.

5. J–N

   Images show a wild‐type plant, CDKA;1‐DE, and the double mutant cdkb1;1 cdkb1;2 (from left to right) on the indicated day after germination and the indicated drug treatment. Scale bars: 1 cm.

Data information: One or two asterisks indicate significant differences within a 5 and 1% confidence interval, respectively (Student's t‐test). Three biological replicates, each containing at least 15 plants, were analyzed. The mean of the root length of each individual experiment was determined and again averaged for the three biological replicates. Graphs represent mean ± SD.

Figure EV7. The triple mutant cycb1;1 cdkb1;1 cdkb1;2 shows no additive effect in DNA damage sensitivity

1. The wild type, the cycb1;1 single mutant, the cdkb1;1 cdkb1;2 double mutant, and the cycb1;1 cdkb1;1 cdkb1;2 triple mutant were grown on control plates without genotoxins and plates supplemented with 1 mM hydroxyurea (HU) or 0.6 μg/ml bleomycin (BLM). Root lengths were measured 10 days after germination.

2. The wild type, the cycb1;1 single mutant, the cdkb1;1 cdkb1;2 double mutant, and the cycb1;1 cdkb1;1 cdkb1;2 triple mutant germinated on control plates and were transferred to plates supplemented with 30 μM cisplatin 3 days after germination. Root lengths were measured 3 days after transfer and the net root growth of 3 days is shown in the graphs.

3. Images show the wild type, the cycb1;1 single mutant, the cdkb1;1 cdkb1;2 double mutant, and the cycb1;1 cdkb1;1 cdkb1;2 triple mutant (from left to right) 6 days after germination (= 3 days after transfer to 30 μM cisplatin plates).

Data information: One or two asterisks indicate significant differences within a 5 and 1% confidence interval, respectively (Student's t‐test). Scale bars: 1 cm. Three biological replicates, each containing at least 15 plants, were analyzed. The mean of the root length of each individual experiment was determined and again averaged for the three biological replicates. Graphs represent mean ± SD.

Figure 5. The CDKB1;1‐CYCB1;1 complex phosphorylates RAD51

1. WT and rad51 mutants were grown on control plates or transferred to plates supplemented with 15 or 30 μM cisplatin, respectively, 3 days after germination. Root lengths were measured 3 days after transfer and the net root growth of 3 days is shown in the graph. Asterisk indicates significant differences within a 5% confidence interval (Student's t‐test). Three biological replicates, each containing at least 15 plants, were analyzed. The mean of the root length of each individual experiment was determined and again averaged for the three biological replicates. Graphs represent mean ± SD.

2. Image shows a wild‐type plant (left) and rad51 mutant (right) grown on control plates. Images were taken 6 days after germination. Scale bar: 1 cm.

3. Image shows a wild‐type plant (left) and rad51 mutant (right) germinated on control plates and transferred to plates supplemented with 15 μM cisplatin 3 days after germination. Images were taken 6 days after germination, that is, 3 days after transfer to cisplatin. Scale bar: 1 cm.

4. Immunostaining of RAD51 foci in the wild‐type and indicated mutant cells after 2 h of treatment with 50 μM cisplatin.

5. Counted numbers of RAD51 foci per cell detected after 2 h of treatment with 50 μM cisplatin in wild‐type and mutant plants. For each sample, the RAD51 foci of 100 cells were counted and grouped into six categories: cells with 0, 1, 2, 3, 4, or > 5 foci per cell.

6. In vitro kinase assay of purified CDK complexes phosphorylating RAD51. RAD51 and histone H1 kinase assays were performed with [γ‐³²P]ATP as a phosphate donor. Proteins were subjected to SDS–PAGE after the kinase reaction and stained with Coomassie Brilliant Blue demonstrating the equal loading of the substrates (lower panels). Phosphorylated proteins were detected by autoradiography (upper panels). The reactions were normalized by using equal amounts of CDKs assuring equal levels of active CDK‐cyclin complexes; the protein blot indicates the relative amounts of the CDKs in the reaction (bottom panel). Abbreviation: p‐RAD51 and p‐histone H1 for [³²P]‐phosphorylated MBP‐RAD51‐His6 and recombinant human histone H1, respectively, resulting from kinase assays with radiolabeled ATP. Asterisks indicate varying amounts of cyclins that can be in the reaction due to purification procedure. 1: without kinase, 2: CDKA;1‐CYCA2;3, 3: CDKB1;1‐CYCA2;3, 4: CDKB1;1‐CYCD2;1, 5: CDKA;1‐CYCB1;1, 6: CDKB1;1‐CYCB1;1.

Figure 6. CYCB1;1 is upregulated after cisplatin treatment

1. Transgenic Arabidopsis plants harboring the CYCB1;1 promoter and a GFP fused to the N‐terminal part of CYCB1;1 including the destruction box grown on control plates and imaged 5 days after germination.

2. Transgenic Arabidopsis plants harboring the CYCB1;2 promoter and a GFP fused to the N‐terminal part of CYCB1;2 including the destruction box grown on control plates and imaged 5 days after germination.

3. Transgenic Arabidopsis plants harboring CDKB1;1 promoter fused to GUS grown on control plates and were stained and imaged 5 days after germination.

4. Transgenic Arabidopsis plants harboring the CYCB1;1 promoter and a GFP fused to the N‐terminal part of CYCB1;1 including the destruction box germinated on control plates and were transferred 4 days after germination to plates supplemented with 50 μM cisplatin and were imaged 24 h after drug application.

5. Transgenic Arabidopsis plants harboring the CYCB1;2 promoter and a GFP fused to the N‐terminal part of CYCB1;2 including the destruction box germinated on control plates and were transferred 4 days after germination to plates supplemented with 50 μM cisplatin and were imaged 24 h after drug application.

6. Transgenic Arabidopsis plants harboring CDKB1;1 promoter fused to GUS germinated on control plates and were transferred 4 days after germination on plates supplemented with 50 μM cisplatin and were stained and imaged 24 h after drug application.

7. Structure of genes tested by ChIP with an anti‐MYC antibody in PRO _SOG1 : SOG1‐Myc and wild‐type plants. A total of four regions were tested as indicated by arrowheads. Asterisks indicate significant differences within a 5% confidence interval (Student's t‐test).

8. Chromatin immunoprecipitation (ChIP) of wild‐type plants and PRO _SOG1 : SOG1‐Myc lines and anti‐MYC antibody. The promoter region of CYCB1;1 is enriched in SOG1‐Myc after cisplatin treatment. Red arrowheads indicate the primer binding sites for PCR.

Data information: Scale bars: 20 μm. Three biological and three technical replicates were performed. Graphs represent mean ± SD.

Figure EV8. CYCB1;3:GFP and CYCB1;4:GFP are not upregulated after cisplatin treatment

1. Transgenic Arabidopsis plants harboring the CYCB1;3 promoter fused to GFP grown on control plates and imaged 4 days after germination.

2. Transgenic Arabidopsis plants harboring the CYCB1;3 promoter fused to GFP germinated on control plates and were transferred 3 days after germination to plates supplemented with 50 μM cisplatin and were imaged 24 h after drug application.

3. Transgenic Arabidopsis plants harboring the CYCB1;4 promoter fused to GFP grown on control plates and imaged 4 days after germination.

4. Transgenic Arabidopsis plants harboring the CYCB1;4 promoter fused to GFP germinated on control plates and were transferred 3 days after germination to plates supplemented with 50 μM cisplatin and were imaged 24 h after drug application.

Data information: Scale bars: 20 μm.

Figure 7. Mutants of cycb1 ku70 are hypersensitive to BLM: genetic interaction between NHEJ und HR

1. A, B

   The wild type, ku70, the double mutants cycb1;1 ku70, cycb1;2 ku70, and cycb1;1 cycb1;2, and the triple mutant cycb1;1 cycb1;2 ku70 were grown on control plates (A) or plates containing 0.6 μg/ml BLM (B) and root lengths were measured 10 days after germination.

2. C

   Graph represents the ratio of the mean growth rate on 0.6 μg/ml BLM compared to control experiments on plates lacking BLM for the wild type, ku70, cycb1;1 ku70, cycb1;2 ku70, and cycb1;1 cycb1;2, and the triple mutant cycb1;1 cycb1;2 ku70.

3. D, E

   Image shows the wild type, ku70, the double mutants cycb1;1 ku70, cycb1;2 ku70, and cycb1;1 cycb1;2, and the triple mutant cycb1;1 cycb1;2 ku70 (from left to right) grown on control plates (D) or plates containing 0.6 μg/ml BLM (E) 10 days after germination.

Data information: One asterisk indicates significant differences within a 5% confidence interval between the wild type and the mutants (Student's t‐test). Two asterisks represent significant differences within a 5% confidence interval between the wild type and the triple mutant cycb1;1 cycb1;2 ku70 as well as a significant reduction of root growth between ku70 and the triple mutant cycb1;1 cycb1;2 ku70. Scale bars: 1 cm. Three biological replicates, each containing at least 15 plants, were analyzed. The mean of the root length of each individual experiment was determined and again averaged for the three biological replicates. Graphs represent mean ± SD.

Figure 8. Division of labor model of the regulation of cell proliferation and DNA damage response in Arabidopsis

DNA damage, for example induced by chemical mutagens, is followed by the activation of the checkpoint kinase ATM (ataxia‐telangiectasia mutated) that activates the transcription factor SOG1 (SUPPRESSOR OF GAMMA RESPONSE 1). On the one hand, SOG1 represses, directly or indirectly, the expression CDKB2 and possible other CDKs as the major driving force of mitosis to allow a cell time for DNA repair. On the other hand, SOG1 directly binds to the promoter of CYCB1;1 and activates its expression. CYCB1;1 builds an active complex together with CDKB1. This complex phosphorylates the DNA binding protein RAD51 (RADIATION SENSITIVE 1) that gets recruited to the DNA damage site. This cascade is required for HR in S and G2 phases of the cell cycle in meristematic cells. To trigger this response, the action of all four B1‐type cyclins is necessary, possibly by providing a threshold of mitotic CDK activity that then gets amplified through SOG1‐dependent stimulation of CYCB1;1 expression.