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. 2019 Nov 18;47(20):10706-10727.
doi: 10.1093/nar/gkz794.

PP4 Phosphatase Cooperates in Recombinational DNA Repair by Enhancing Double-Strand Break End Resection

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Free PMC article

PP4 Phosphatase Cooperates in Recombinational DNA Repair by Enhancing Double-Strand Break End Resection

María Teresa Villoria et al. Nucleic Acids Res. .
Free PMC article

Abstract

The role of Rad53 in response to a DNA lesion is central for the accurate orchestration of the DNA damage response. Rad53 activation relies on its phosphorylation by Mec1 and its own autophosphorylation in a manner dependent on the adaptor Rad9. While the mechanism behind Rad53 activation has been well documented, less is known about the processes that counteract its activity along the repair of a DNA adduct. Here, we describe that PP4 phosphatase is required to avoid Rad53 hyper-phosphorylation during the repair of a double-strand break, a process that impacts on the phosphorylation status of multiple factors involved in the DNA damage response. PP4-dependent Rad53 dephosphorylation stimulates DNA end resection by relieving the negative effect that Rad9 exerts over the Sgs1/Dna2 exonuclease complex. Consequently, elimination of PP4 activity affects resection and repair by single-strand annealing, defects that are bypassed by reducing Rad53 hyperphosphorylation. These results confirm that Rad53 phosphorylation is controlled by PP4 during the repair of a DNA lesion and demonstrate that the attenuation of its kinase activity during the initial steps of the repair process is essential to efficiently enhance recombinational DNA repair pathways that depend on long-range resection for their success.

Figures

Figure 1.
Figure 1.
Pph3 is required for DNA repair by homologous recombination by controlling multiple components of the DDR. (A) Schematic representation depicting relevant genomic structures of the strain used to assess DNA repair by SSA/BIR. The location of an U2 probe and the restriction endonuclease cleavage sites used for Southern blot analysis to detect the formation of the repair product are shown. Arrow indicates the localization of the DSB. (B) Physical analysis of wild-type and pph3Δ mutant strains carrying the DNA repair system depicted in (A). Cells were grown overnight in YP-Raffinose before adding galactose to induce HO expression. Samples were taken at different time points and genomic DNA was extracted, digested with KpnI and analysed by Southern blot. Blots were hybridized with an U2 and ACT1 (as loading control) DNA probes. Graphs show the mean ± SD of the band signals from three independent experiments. All data were normalized using actin as loading control. Replicates were averaged and statistical significance of differences assessed by a two-tailed unpaired Student's t-test. (C) FACS profiles of samples taken from (B). (D) Samples at 0, 6 and 12 h from the experiment shown in (B) were taken, processed and subjected to mass spectrometry. DDR-related proteins containing at least one phospho-peptide enriched in the absence of PP4 were classified depending on their averaged phosphorylation. Heat map scale represents a log2 fold change over maximum. Yellow, blue and red indicate relative amount of protein phosphorylation (yellow, low; blue, medium; red, high). (E) Samples from the experiment shown in (B) were collected at the indicated time points, proteins TCA extracted and subjected to Western blotting. Coomassie staining is shown as loading control. (F) Schematic representation of S. cerevisiae Rad53 illustrating the SQ/TQ clusters (yellow), forkhead-associated domains (blue), kinase domain (red) and nuclear localization signal (green). An amino acid residue number scale is shown. (G) Graphs representing normalized P-intensities of the Rad53 phospho-peptides identified at 0, 6 and 12 h from the HO induction.
Figure 2.
Figure 2.
PP4 activity is required to stimulate DNA end resection. (A) Schematic representation of the assay used to determine resection efficiency at the MAT locus. The diagram includes the recognition sites for StyI and the localization of the probes used (red lines) for each distance. Table includes the StyI-StyI DNA fragments length for each probe. (B) Exponentially wild-type and pph3Δ cells growing in YP-Raffinose were synchronized in G1 by using the α-factor pheromone and released into fresh media for 1 h. Induction of HO expression was attained by adding galactose to the media and samples were taken at the indicated time points. Proteins were TCA extracted and subjected to Western blotting. Coomassie staining is shown as loading control. (C) FACS analysis of the experiment depicted in (B). (D) Physical analysis by Southern blot of wild-type and pph3Δ strains containing the resection assay described in (A). Samples were taken under the same experimental conditions as in (B), DNA extracted, digested with StyI and blotted. (E) Band intensities from the experiment shown in (D) were measured, normalized against actin and plotted. Graphs represent the mean ± SD from three independent experiments. P-values were calculated using a two-tailed unpaired Student's t-test.
Figure 3.
Figure 3.
Reduction of Rad53 phospho-levels alleviates PP4-dependent defects in DNA end resection and repair by SSA/BIR. (A) Exponentially growing cells from pph3Δ and pph3Δ rad53K227A backgrounds were synchronized in G1 by using the pheromone α-factor and released into fresh media for 1 h. After inducing the HO expression by adding galactose to the media, samples were collected at the indicated time points and processed for Western blotting. Coomassie staining is shown as loading control. (B) Southern blot analysis of samples taken from the experiment shown in (A) to determine resection efficiency. (C) FACS analysis of cells collected from (A). (D) Band signals from the Southern blot depicted in (B) were quantified, normalized against actin and charted. (E) Cells from pph3Δ and pph3Δ rad53K227A strains were exponentially grown overnight in raffinose-containing media before adding galactose. Samples were collected at the indicated time points and subjected to Western blotting. Coomassie staining is shown as loading control. (F) Southern blots of pph3Δ and pph3Δ rad53K227A cells carrying the DNA repair assay illustrated in Figure 1A. Samples from the experiment shown in (E) were taken at the indicated time points, DNA extracted, digested with KpnI and blotted. Blots were probed with an U2 DNA sequence and ACT1 as loading control. Graphs representing the quantification of the band signals from the Southern blot experiment relative to the actin control. (G) FACS profile for DNA content of samples collected from the experiment assayed in (F). All graphs in the figure represent the mean ± SD from three independent experiments. P-values were calculated using a two-tailed unpaired Student's t-test.
Figure 4.
Figure 4.
PP4's function in DNA repair becomes essential when repairing by SSA. (A) rad51Δ and rad51Δ pph3Δ cells were grown overnight in raffinose-containing media and supplemented with galactose to induce the expression of the HO. Samples were collected at the indicated time points, proteins TCA extracted and subjected to Western blotting. Coomassie staining is shown as loading control. (B) Southern blot analysis of rad51Δ and rad51Δ pph3Δ strains carrying the DNA repair system depicted in Figure 1A. Samples from the experiment shown in (A) were taken at the indicated time points, genomic DNA extracted, digested with KpnI and analysed by Southern blot. Blots were hybridized with an U2 and ACT1 (as loading control) DNA probes. Graphs represent the quantification of the band signals detected in the Southern blot. (C) FACS analysis of cells collected from (B). (D) rad51Δ and rad51Δ pph3Δ cells were grown overnight in YP-Raffinose and supplemented with galactose. Samples were taken at different intervals, genomic DNA extracted, digested with StyI and analysed by Southern blot. Blots were hybridized with probes located at 10 kb and 17 kb upstream the HO cut site. Graphs represent the quantification of the band signals detected in the Southern blot. (E) Cells from rad51Δ pph3Δ and rad51Δ pph3Δ rad53K227A background strains were grown overnight in media containing raffinose before adding galactose. Samples were collected at the indicated time points and subjected to Western blotting. Coomassie staining is shown as loading control. (F) Southern blot of rad51Δ pph3Δ and rad51Δ pph3Δ rad53K227A cells carrying the DNA repair system depicted in Figure 1A. Samples from the experiment shown in (E) were taken at the indicated time points, DNA extracted, digested with KpnI and blotted. Blots were hybridized with an U2 and ACT1 (as loading control) DNA probes. Graphs represent the quantification of the band signals detected in the Southern blot. (G) FACS profile for DNA content of samples collected in (F). All graphs in the figure represent the mean ± SD of the band signals from three independent Southern blot experiments. All data were normalized to actin. Replicates were averaged and statistical significance of differences assessed by a two-tailed unpaired Student's t-test.
Figure 5.
Figure 5.
PP4's role in DNA end resection is mainly driven by the modulation of the Sgs1/Dna2 pathway. (A) Cultures of pph3Δ, exo1Δ and exo1Δ pph3Δ cells were grown in YP-Raffinose, blocked in G1 and released into fresh media containing galactose. Cells were collected at the indicated time points and processed for FACS. (B) Resection assay by Southern blot of samples taken from (A). Cells were DNA extracted, digested with StyI and blotted. (C) The density of the bands depicted in (B) was measured, normalized to actin and plotted. Graphs represent the mean ± SD from three independent experiments. P-values were calculated using a two-tailed unpaired Student's t-test. Asterisk denotes statistical significance between pph3Δ and exo1Δ strains. Hash denotes statistical significance between exo1Δ and exo1Δ pph3Δ strains. (D) Cultures of pph3Δ, sgs1Δ and sgs1Δ pph3Δ cells were cultured in raffinose containing media, arrested in G1 and released into fresh media containing galactose. A FACS profile of cells collected at the indicated time points is represented. (E) Resection analysis by Southern blot of samples taken from (D). Cells were DNA extracted, digested with StyI and blotted. (F) The intensity of the bands depicted in (E) was measured, normalized to actin and charted. Graphs represent the mean ± SD from three independent experiments. P-values were calculated using a two-tailed unpaired Student's t-test. Asterisk denotes statistical significance between pph3Δ and sgs1Δ strains. Hash denotes statistical significance between sgs1Δ and sgs1Δ pph3Δ strains.
Figure 6.
Figure 6.
PP4 modulates Rad9 by acting over Rad53. (A) Schematic representation of S. cerevisiae Rad9 illustrating the Chk1 activating domain (orange), Mec1 serine cluster domain (red), DNA interaction TUDOR domain (yellow) and protein-protein recognition module BRCT domain (blue). An amino acid residue number scale is shown. (B) Graphs representing normalized P-intensities of the Rad9 phospho-peptides identified by mass spectrometry at 0, 6 and 12 h from the HO induction. (C) Wild-type, pph3Δ, rad53K227A and pph3Δ rad53K227A cells containing the endogenous Rad9 tagged with the HA epitope were subjected to α-factor block and released in the presence of galactose. Samples were blotted using anti-HA antibodies. Coomassie blue staining is depicted as loading control. (D) ChIP analysis of Rad9 binding around the HO-induced DSB at the MAT locus in wild-type, pph3Δ, pph3Δ rad53K227A and rad53K227A cells. Galactose was added to asynchronous cell cultures and samples were taken at 1 h and 2 h. Each time point was normalized to the input signal. Graphs represent fold enrichment relative to the non-antibody negative control. Binding at the ACT1 locus on chromosome VI is shown for comparison. Graphs represent the mean ± SD from three independent experiments.
Figure 7.
Figure 7.
Disruption of Rad9 bypasses the resection and SSA repair defects observed in the absence of PP4 activity. (A) Exponentially growing cells of pph3Δ and pph3Δ rad9Δ strains were synchronized in G1 by using α-factor and released into fresh media for 1 h. After inducing the HO expression by adding galactose to the media samples were collected at the indicated time points and subjected to a resection analysis by Southern blotting. (B) FACS profile of cells collected from (A). (C) Samples from (A) were taken at the indicated time points and subjected to Western blotting. Coomassie staining is shown as loading control. (D) Bands from the Southern blot depicted in (A) were quantified, normalized against actin and plotted. (E) Physical analysis of pph3Δ and pph3Δ rad9Δ cells harbouring the DNA repair assay portrayed in Figure 1A. Cells were grown in YP-Raffinose and transferred to media supplemented with galactose. Samples were collected at different time points, genomic DNA extracted, digested with KpnI and analysed by Southern blot. Blots were hybridized with an U2 and ACT1 (as loading control) DNA probes. Graphs show the quantification of the band signals relative to actin. (F) FACS profile for DNA content of samples taken from (E). (G) Samples from the experiment shown in (E) were collected at the indicated time points, TCA extracted and subjected to Western blotting. Coomassie staining is shown as loading control. (H) Physical analysis of rad51Δ pph3Δ and rad51Δ pph3Δ rad9Δ backgrounds harbouring the DNA repair assay portrayed in Figure 1A. Cells were grown overnight in YP-Raffinose and supplemented with galactose. Samples were taken at different time points, genomic DNA extracted, digested with KpnI and analysed by Southern blot. Blots were hybridized with an U2 and ACT1 (as loading control) DNA probes. Graphs represent the quantification of the band signals relative to actin. (I) FACS profile for DNA content of samples collected from (H). (J) Samples from the experiment shown in (H) were collected at the indicated time points, TCA extracted and subjected to Western blotting. Coomassie staining is shown as loading control. All graphs in the figure represent the mean ± SD from three independent experiments. Replicates were averaged and statistical significance of differences assessed by a two-tailed unpaired Student's t-test.
Figure 8.
Figure 8.
Elimination of H2A phosphorylation at Ser129 rescues both DNA end resection and repair defects of cells lacking PP4 activity. (A) ChIP analysis of Rad9 binding around the HO-induced DSB at the LEU2 locus in wild-type, pph3Δ, and pph3Δhta1/hta2-S129* cells. Each time point was normalized to the input signal. Graphs represent fold enrichment relative to the non-antibody negative control. (B) YP-Raffinose cell cultures of YMV80 derivative pph3Δ and pph3Δ hta1/hta2-S129* cells were supplemented with galactose to induce the HO expression and samples were collected at the indicated time points. Proteins were TCA extracted and subjected to Western blotting. Coomassie staining is shown as loading control. (C) Physical analysis of pph3Δ and pph3Δhta1/hta2-S129* cells harbouring the DNA repair assay portrayed in Figure 1A. Samples from the experiment shown in (B) were taken at different time points, genomic DNA extracted, digested with KpnI (DNA repair) or StyI (resection) and analysed by Southern blot. Blots were hybridized with an U2 probe to determine DNA repair efficiency (top panel) and with probes located at 10 kb and 17 kb upstream the HO cut site for the analysis of resection (bottom panel). In both cases ACT1 was used as loading control. (D) Graphs represent the quantification of the donor and product band signals obtained from the Southern blot experiment shown in (C, top panel) relative to the actin signal. (E) FACS profile for DNA content of samples collected in (C). (F) Graphs represent the quantification of the 10r and 17r band signals obtained from the Southern blot experiment shown in (C, bottom panel) relative to the actin signal. (G) Model to integrate the role of PP4 in DSB repair. All graphs in the figure represent the mean ± SD from three independent experiments. Replicates were averaged and statistical significance of differences assessed by a two-tailed unpaired Student's t-test.

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