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. 2010 Mar 1;24(5):502-15.
doi: 10.1101/gad.1869110.

De Novo Telomere Formation Is Suppressed by the Mec1-dependent Inhibition of Cdc13 Accumulation at DNA Breaks

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Free PMC article

De Novo Telomere Formation Is Suppressed by the Mec1-dependent Inhibition of Cdc13 Accumulation at DNA Breaks

Wei Zhang et al. Genes Dev. .
Free PMC article

Abstract

DNA double-strand breaks (DSBs) are a threat to cell survival and genome integrity. In addition to canonical DNA repair systems, DSBs can be converted to telomeres by telomerase. This process, herein termed telomere healing, endangers genome stability, since it usually results in chromosome arm loss. Therefore, cells possess mechanisms that prevent the untimely action of telomerase on DSBs. Here we report that Mec1, the ATR ortholog, couples the detection of DNA ends with the inhibition of telomerase. Mec1 inhibits telomere healing by phosphorylating Cdc13 on its S306 residue, a phosphorylation event that suppresses Cdc13 accumulation at DSBs. Conversely, telomere addition at accidental breaks is promoted by Pph3, the yeast protein phosphatase 4 (PP4). Pph3 is itself modulated by Rrd1, an activator of PP2A family phosphatases. Rrd1 and Pph3 oppose Cdc13 S306 phosphorylation and are necessary for the efficient accumulation of Cdc13 at DNA breaks. These studies therefore identify a mechanism by which the ATR family of kinases enforces genome integrity, and a process that underscores the contribution of Cdc13 to the fate of DNA ends.

Figures

Figure 1.
Figure 1.
A genetic screen identifies Rrd1 as a factor important for de novo telomere formation. (A) Schematic representation of the Chr V GCR assay. The test strain (derivative of hxt13∷URA3) possesses two counterselectable markers (CAN1 and URA3) located near the telomeric end of Chr V–L. GCR events are identified by the simultaneous loss of CAN1 and URA3, which confer resistance to canavanine (Can) and 5-FOA (FOA). PIF1 inhibits telomere healing, and its deletion skews GCR events to the telomere healing pathway. (B) An outline of the transposon-based genetic screen carried out in this study. (C) Identification of telomere healing mutants by the papillation assay. Photographs were taken from the primary screen data. The top two panels show the papillation phenotypes of the control pif1Δ and pif1Δ yku70Δ strains, respectively. The bottom left panel shows a strain, later identified as a transposon disrupting the RRD1 gene. The bottom right panel displays a typical uncharacterized strain that scored negative by displaying a similar number of colonies as the pif1Δ control. (D) Quantitation of spontaneous GCR rates using the Chr V assay. The strains tested were derivatives of hxt13∷URA3 (WT), and, in the same background, pif1Δ, pif1Δ yku70Δ, pif1Δ cik1Δ, pif1Δ rts1Δ, pif1Δ irc6Δ, pif1Δ pex10Δ, pif1Δ ctf18Δ, and pif1Δ rrd1Δ. The data were obtained following a minimum of two independent fluctuation tests with 11 cultures. The data are presented as the mean ± SEM. (E) Quantitation of spontaneous GCR rates using the Chr V assay. The strains tested were derivatives of hxt13∷URA3 (WT), and, in the same background, pif1-m2, pif1-m2 yku70Δ, pif1-m2 yku80Δ, pif1-m2 rrd1Δ, and pif1-m2 yku80Δ rrd1Δ. The data were obtained following a minimum of two independent fluctuation tests with 11 cultures. The data are presented as the mean ± SEM. (F) Quantitation of MMS-induced frequency of GCR events using the Chr V assay. The strains tested were derivatives of hxt13∷URA3 (WT), and, in the same background, sul2Δ, yku70Δ, rrd1Δ, rrd1Δ <pRS414>, and rrd1Δ <pRRD1>. pRRD1 harbors the RRD1 gene under the control of its own promoter, in pRS414. The data are presented as the mean ± SEM (N = 3).
Figure 2.
Figure 2.
Rrd1 facilitates telomere healing at DSBs with no or short TG1–3 tracts. (A) rrd1Δ cells maintain normal telomere length. The TRFs of the indicated strains were detected by Southern blot with a Y′-TG probe targeting telomere sequences. (B) Schematic representation of the Chr V–L. The breakpoints of the GCR events must land between the essential gene PCM1 (blue) and the selection markers CAN1 and URA3 (yellow). Positions of independent telomere additions or translocations are indicated for wild-type (below) and rrd1Δ (above) strains. (C) Distribution of the number of TG1–3 residues found at the breakpoints of the telomere healing events described in B. (D) Breakpoint junction sites from telomere addition events recovered from wild-type or rrd1Δ strains. The events shown correspond to telomere addition events that occurred outside the NPR2 TG-rich hot spot. Nucleotide sequences are shown as 5′-to-3′ direction, and telomere repeat polymerization began following the last nucleotide indicated. Nucleotides conforming to a TG1–3 sequence are capitalized.
Figure 3.
Figure 3.
A TGn-HO set of strains to study telomere healing at HO-induced DSBs. (A) Schematic representation of the modified Chr VII–L to generate the TGn-HO set of strains. The ADH4 locus was replaced by the ADE2 or URA3 gene followed by telomere seed sequences of different sizes (no telomeric seed, 5, 11, 17, and 81 bp of TG1–3 repeats), followed by the HO endonuclease cleavage site. The location of the probe used for Southern blotting (star on a bar) is also shown. (B) Quantitation of telomere addition frequency, as determnined by αAA resistance. The strains tested were PIF1 (WT; black bars) and pif1-m2 (gray bars) derivatives of strains TG0-HO, TG5-HO, TG11-HO, TG17-HO, and TG81-HO. The data are presented as the mean ± SEM (N ≥ 3). (C) The pif1-m2 mutation stimulates telomere addition in the TG11-HO strain. Cultures of wild-type and pif1-m2 derivatives of TG11-HO rad52Δ were arrested with nocodazole. HO expression was induced using galactose, and samples were taken at the indicated time points. In addition, Lys+ and Lys derivatives of TG11-HO rad52Δ pif1-m2 were collected from the genetic assay based on αAA selection. A Southern blot analysis of EcoRV-cut genomic DNA probed with a section of the URA3 gene is shown. The band labeled PRE represents the EcoRV-digested fragment from Chr VII–L. After cleavage with HO, this fragment is converted into a new band (CUT). The URA3 probe also detects the ura3-52 locus (INT) and serves as a loading control. New telomere elongation forms a smear above the CUT band. (D–F) Quantitation of telomere addition frequency, as determnined by αAA resistance. The strains tested were wild-type (WT), rrd1Δ, and est2Δ derivatives of pif1-m2 TG0-HO (D), pif1-m2 TG5-HO (E), or TG11-HO (F). The data are presented as the mean ± SEM (N ≥ 3). (G) Quantitation of telomere addition frequency of wild-type, rrd1Δ, yku80Δ, rrd1Δ yku80Δ, and est2Δ derivatives of pif1-m2 TG0-HO. The data are presented as the mean ± SEM (N ≥ 3). (H) Quantitation of telomere addition frequency of wild-type, rrd1Δ, yku80Δ, and rrd1Δ yku80Δ derivatives of TG0-HO. The data are presented as the mean ± SEM (N ≥ 3).
Figure 4.
Figure 4.
Mec1 and Rrd1–Pph3 form a phosphoregulatory circuit that controls telomere healing at DSBs. (A) Model of Rrd1 action, given its role as a PP2A-type phosphatase activator. Rrd1 might activate a phosphatase that antagonizes a phosphorylation event that inhibits telomere healing. (B) Quantitation of spontaneous GCR rates using the Chr V assay. The strains tested were derivatives of hxt13∷URA3 (WT), and, in the same background, pif1Δ, pif1Δ sul2Δ, pif1Δ, pif1Δ rrd1Δ, pif1Δ pph3Δ, pif1Δ ppg1Δ, pif1Δ sit4Δ, and pif1Δ rrd1Δ pph3Δ. The data were obtained following a minimum of two independent fluctuation tests with 11 cultures. The data are presented as the mean ± SEM. (C) Quantitation of telomere addition frequency of wild-type, ppg1Δ, rrd1Δ, pph3Δ, and rrd1Δ pph3Δ derivatives of pif1-m2 TG5-HO. The data are presented as the mean ± SEM (N ≥ 3). (D) Quantitation of telomere addition frequency of wild-type and mec1Δ derivatives of pif1-m2 sml1Δ TG0-HO (left panel), and wild-type, tel1Δ, and rad9Δ derivatives of pif1-m2 TG0-HO (right panel). SML1 was deleted to maintain the viability of mec1Δ. The data are presented as the mean ± SEM (N ≥ 3). (E) Quantitation of telomere addition frequency of wild-type and mec1Δ derivatives of pif1-m2 sml1Δ TG5-HO. The telomere addition frequency of mec1Δ derivatives containing plasmids that expressed either MEC1 or a mec1 kinase-dead allele (mec1-kd) is also shown. The data are presented as the mean ± SEM (N ≥ 3). (F) Quantitation of telomere addition frequency of wild-type, mec1Δ, rrd1Δ, and mec1Δ rrd1Δ derivatives of pif1-m2 sml1Δ TG5-HO. The data are presented as the mean ± SEM (N ≥ 3). (G) Quantitation of telomere addition frequency of wild-type, mec1Δ, rrd1Δ, sml1Δ, and mec1Δ sml1Δ derivatives of pif1-m2 TG11-HO. The data are presented as the mean ± SEM (N ≥ 3).
Figure 5.
Figure 5.
Tethering Cdc13 to a DSB overrides the Rrd1/Pph3-Mec1 regulatory network. (A) Schematic representation of the modified Chr VII–L in the GBD fusion assay. The restriction sites (EcoRV) and the probe location (star on a bar) used for Southern blotting are depicted. Telomere healing events stimulated by the GBD fusion can be detected via the loss of LYS2 and the retention of ADE2. (B) Quantitation of telomere addition frequency of wild-type (WT), yku80Δ, rrd1Δ, and rrd1Δ aur1∷RRD1 derivatives of the GAL4UAS rad52Δ strain in the presence of either an empty GBD-expressing plasmid (−) or the GBD-Est1 fusion (Est1). The data are presented as the mean ± SEM (N ≥ 3). (C) Wild-type and rrd1Δ cells were grown in galactose to induce HO expression, and samples were taken at the indicated time points. A Southern blot of EcoRV-cut genomic DNA was probed with a portion of the ADE2 gene. The band labeled PRE represents the EcoRV restriction-digested fragment from Chr VII–L. After HO cleavage, this fragment is converted into a new band (CUT). New telomere elongation appears as a smear above the CUT band. (D) Quantitation of telomere addition frequency of wild-type and mec1Δ derivatives of a GAL4UAS sml1Δ rad52Δ strain (black bars), and wild-type and rrd1Δ derivatives of a GAL4UAS rad52Δ strain (gray bars). All strains expressed the GBD-Est1 fusion. The experiments were carried out in nocodazole-arrested cells. The data are presented as the mean ± SEM (N ≥ 3). (E) Quantitation of telomere addition frequency of wild-type, yku80Δ, and rrd1Δ derivatives of a GAL4UAS rad52Δ strain expressing the GBD-Cdc13RD fusion. As a control, we also determined the telomere addition frequency of a GAL4UAS rad52Δ strain expressing the nonfunctional GBD-Cdc13RD-est fusion. The data are presented as the mean ± SEM (N ≥ 3). (F) Quantitation of telomere addition frequency of wild-type and mec1Δ derivatives of a GAL4UAS sml1Δ rad52Δ strain (black bars), and wild-type and rrd1Δ derivatives of a GAL4UAS rad52Δ strain (gray bars). All strains expressed the GBD-Cdc13RD fusion. The experiments were carried out in nocodazole-arrested cells. The data are presented as the mean ± SEM (N ≥ 3).
Figure 6.
Figure 6.
Cdc13 S306 phosphorylation by Mec1 inhibits telomere healing. (A) Western blot analysis of whole-cell extracts obtained from CDC13-Myc, cdc13-S306A-Myc, and wild-type (WT) strains, before and after DNA damage (zeocin) treatment. In the top panel, the blot was probed with the Cdc13 pS306 antibody. In the second panel, the blot was probed with a monoclonal antibody against the Myc epitope. The third panel was probed with a Rad53 antibody to control for the addition of zeocin. (Bottom panel) Pgk1 was used as a loading control. (B) Western blot analysis of whole-cell extracts obtained from sml1Δ, sml1Δ tel1Δ, sml1Δ mec1Δ, and sml1Δ mec1Δ tel1Δ strains, before and after DNA damage (zeocin) treatment. In the top panel, the blot was probed with the Cdc13 pS306 antibody. (Middle panel) Rad53 phosphorylation was used as a control for the addition of zeocin and Mec1 activity. (Bottom panel) Pgk1 was used as a loading control. (C) Quantitation of telomere addition frequency of wild-type (WT), cdc13-S306A, cdc13-S306E, rrd1Δ, cdc13-S306A rrd1Δ, sml1Δ, cdc13-S306A sml1Δ, mec1Δ sml1Δ, and cdc13-S306A mec1Δ sml1Δ derivatives of pif1-m2 TG5-HO. The data are presented as the mean ± SEM (N ≥ 3).
Figure 7.
Figure 7.
(A) ChIP of Cdc13-Myc to the MAT-HO locus in wild-type (WT), cdc13-S306A, rrd1Δ, and cdc13-S306A rrd1Δ derivatives of the JKM179 donorless strain. Relative enrichment at the HO locus (MAT 1 kb and 2.5 kb) over the negative control locus PDI1 is shown. The data are presented as the mean ± SEM (N = 3). (B) Cdc13 pS306 phosphatase assays with purified Pph3-TAP and Sit4-TAP. Detailed descriptions of the purification and assay are found in Supplemental Figure S9. After reactions, peptides were immobilized by slot-blotting, and the biotinylated peptides were detected using extravidin (EA) coupled to HRP. The eluate from an untagged strain was used as negative control. Loss of signal in the bound fraction indicates phosphatase activity. (C) ChIP of Pph3-Myc to the MAT-HO locus in the JKM179 donorless strain. Relative enrichment at the HO locus (MAT 1 kb and 2.5 kb) over the negative control locus PDI1 is shown. The data are presented as the mean ± SEM (N = 3). (D) Quantitation of spontaneous GCR rates using the Chr V assay. The strains tested were derivatives of hxt13∷URA3 (WT), and, in the same background, cdc13-S306A and pif1-4A. The data are presented as the mean ± SEM. (E) Quantitation of telomere addition frequency of wild-type (WT), cdc13-S306A, and Pif1-4A derivatives of TG5-HO. The data are presented as the mean ± SEM (N ≥ 3). (F) Model of the regulation of de novo telomere formation by Mec1 and Rrd1–Pph3. (Left panel) At DSBs that contain limited telomere-like seed sequences, ssDNA-bound Mec1 phosphorylates Cdc13 on its S306 residue to suppress Cdc13 accumulation at resected ends, possibly via the inhibition of a protein–protein interaction. (Right panel) However, in situations where a DSB is irreparable, cells can allow telomerase to act on a DSB substrate when all other DNA repair options have been exhausted. In this situation, the accumulation of Pph3 at the DSB site allows it to dephosphorylate Cdc13 locally in order to allow its recruitment to the break, which in turn promotes telomerase action. The new telomere is then masked from the DSB repair and signaling machinery, and promotes recovery from the checkpoint arrest.

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