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. 2001 Nov 1;15(21):2809-21.
doi: 10.1101/gad.903501.

Two checkpoint complexes are independently recruited to sites of DNA damage in vivo

J A Melo  1 , J Cohen, D P Toczyski
Affiliations

Two checkpoint complexes are independently recruited to sites of DNA damage in vivo

J A Melo et al. Genes Dev. .

Abstract

The Ddc1/Rad17/Mec3 complex and Rad24 are DNA damage checkpoint components with limited homology to replication factors PCNA and RF-C, respectively, suggesting that these factors promote checkpoint activation by "sensing" DNA damage directly. Mec1 kinase, however, phosphorylates the checkpoint protein Ddc2 in response to damage in the absence of all other known checkpoint proteins, suggesting instead that Mec1 and/or Ddc2 may act as the initial sensors of DNA damage. In this paper, we show that Ddc1 or Ddc2 fused to GFP localizes to a single subnuclear focus following an endonucleolytic break. Other forms of damage result in a greater number of Ddc1-GFP or Ddc2-GFP foci, in correlation with the number of damage sites generated, indicating that Ddc1 and Ddc2 are both recruited to sites of DNA damage. Interestingly, Ddc2 localization is severely abrogated in mec1 cells but requires no other known checkpoint genes, whereas Ddc1 localization requires Rad17, Mec3, and Rad24, but not Mec1. Therefore, Ddc1 and Ddc2 recognize DNA damage by independent mechanisms. These data support a model in which assembly of multiple checkpoint complexes at DNA damage sites stimulates checkpoint activation. Further, we show that although Ddc1 remains strongly localized following checkpoint adaptation, many nuclei contain only dim foci of Ddc2-GFP, suggesting that Ddc2 localization may be down-regulated during resumption of cell division. Lastly, visualization of checkpoint proteins localized to damage sites serves as a useful tool for analysis of DNA damage in living cells.

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Figures

Figure 1
Figure 1
Ddc1 forms a single subnuclear focus after a dsDNA break. (a) Southern blot of DNA proximal to the HO site at the telomere of chromosome VII. To monitor the rate of HO-induced cleavage, log-phase cells were transferred from raffinose to galactose media to induce the HO endonuclease and samples were collected at time points indicated. The uncleaved band is heterogeneous in size because it contains telomeric sequences. The cleaved fragment disappears at late time points because of gradual degradation. (b) Visualization of Ddc1–GFP. Cells at the zero time point were grown in glucose media to repress HO expression; cells at 3-, 6-, 12-, and 16-h time points were grown in raffinose media, then transferred to galactose media for the indicated time. Inset at t = 0 representative of occasional Ddc1–GFP foci in HO-uninduced cells. At the 12-h and 16-h time points, multiple focal planes were merged to demonstrate that Ddc1–GFP foci are seen in all nuclei. The arrowheads in the 12-h time point indicate the presence of an adapted anaphase cell containing a single focus in each nucleus. (c) Fraction of cells containing Ddc1–GFP foci as a time course of HO induction. Data combined from all focal planes within a field. (d) Time course of adaptation following an HO break. Cells were grown in raffinose media, transferred to galactose media for 2 h, sonicated, and plated to raffinose plus galactose plates. Microcolonies containing more than one cell (>2 cell bodies) were considered adapted.
Figure 2
Figure 2
Ddc1 forms multiple nuclear foci following cdc13-1-induced and cdc9-1-induced DNA damage. Log phase cultures of cdc13-1 DDC1–GFP and cdc9-1 DDC1–GFP cells were shifted to 34°C and 36°C, respectively, and visualized after 2 h. Also shown are asynchronously growing wild-type control cells containing DDC1–GFP alone after shift to 34°C for 2 h.
Figure 3
Figure 3
Ddc1 requires Rad24, Rad17, and Mec3 for damage-inducible localization. cdc13-1 DDC1–GFP cells deleted for RAD17, MEC3, RAD24, MEC1, DDC2, RAD53, or RAD9 were raised to 34°C for 2 h before visualization of GFP. Single focal planes for each sample are shown.
Figure 4
Figure 4
Localization of other checkpoint proteins fused to GFP in cdc13-1-damaged cells. GFP fusions generated for Rad53, Rad9, and Ddc2 were expressed under the endogenous promotor for each protein in cdc13-1 cells. Log phase cultures were shifted to 34°C for 2 h before visualization of GFP fusions. Arrowheads indicate the presence of occasional faint foci in cells expressing Rad53–GFP and Rad9–GFP.
Figure 5
Figure 5
Ddc2–GFP forms a single focus in HO break-induced cells. (a) Fraction of cells containing Ddc2–GFP foci as a time course of HO induction. Data combined from all focal planes within a field. (b) Visualization of Ddc2–GFP foci following an HO break. HO endonuclease was induced in cells as described in Figure 1. Cells grown in glucose to repress HO expression are presented as the t = 0 time point. A representative focal plane for 0, 3, 6, 12, and 16 h after HO induction is shown. (c) Chromatin immunoprecipation of Ddc2–GFP at an HO break site. The t = 0 represents cells grown in glucose to repress HO induction. Cells collected at each indicated time point were fixed and analyzed by chromatin IP. Ddc2–GFP and cross-linked DNA were immunoprecipitated using affinity-purified anti-GFP antibodies. After reversal of cross-linking and DNA purification, PCR was performed using HO-2 primers directed to unique DNA sequence 0.4 kb from the HO cleavage site. Primers to amplify unlinked TUB1 sequence were used as an internal control in each PCR reaction. Input DNA was isolated from fixed cells at each time point before immunoprecipitation, and treated in parallel with IP samples in preparation for PCR. Control strains deleted for DDC1 or MEC1 or lacking a Ddc2–GFP tag were grown and harvested at the 7-h time point and treated in parallel to the wild-type Ddc2–GFP strain. (d) The HO-2 and TUB1 bands in c were quantitated and the ratio of HO-2:TUB1 band intensities calculated for each IP and input DNA sample. Fold enrichment was calculated as the ratio of HO-2:TUB1 in the IP samples to its respective input DNA control and graphed as shown.
Figure 6
Figure 6
MEC1 is required for Ddc2 localization following cdc13-1-induced damage. Log phase cdc13-1 DDC2–GFP cells deleted for DDC1, RAD17, MEC3, RAD24, MEC1, RAD53, or RAD9 were raised to 35°C for 2 h before visualization of GFP. Ddc2–GFP localization in CDC13 checkpoint-proficient cells at 35°C is shown at bottom right. Single focal planes for each sample are shown.
Figure 7
Figure 7
Ddc1 localization shows that the two ends of an intrachromosomal DSB remain associated, and that cells lacking Rad52 contain high levels of unrepaired damage. (a) Cells containing an HO site near the chromosome VII SRM1 locus were grown in raffinose, transferred to galactose media, and Ddc1–GFP was visualized after 6 h. (b) Log-phase rad52 cells in the absence of induced DNA damage were visualized for Ddc1–GFP.
Figure 8
Figure 8
The substrate recruitment model: Checkpoint activation by the concentration of checkpoint factors on a DNA strand. Mec1/Ddc2 and the Ddc1/Mec3/Rad17 complex are recruited to DNA damage independently, promoting their interaction. Multiple Ddc1/Rad17/Mec3 complexes are loaded by the Rad24/RF-C complex and may function to recruit the substrates of Mec1.
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