1. Volume 14, Issue 4, Pages 515-522 (May 2004)
Reciprocal Association of the Budding Yeast ATM-Related Proteins Tel1 and Mec1 with Telomeres In Vivo 
Hideki Takata, Yutaka Kanoh, Norio Gunge, Katsuhiko Shirahige, Akira Matsuura 
Molecular Cell 
Volume 14, Issue 4, Pages (May 2004)
DOI: /S (04)00262-X

2. Figure 1
Specific Association of Tel1p and Mec1p with Telomeres In Vivo
(A) The 18 myc-tagged Mec1p and 5 HA-tagged Tel1p proteins are fully functional. Left: Genomic DNA isolated from BY4741 (MEC1 TEL1), LSS36 (myc-MEC1 TEL1), and LSS65 (myc-MEC1 HA-TEL1) was digested with XhoI, followed by Southern blot analysis using the subtelomeric Y′ fragment as the probe. Middle: The cells were grown in liquid YPD medium and diluted serially, and 5 μl aliquots of each dilution were spotted onto YPD with or without 150 mM hydroxyurea (HU). In the bottom row, myc-mec1-KD cells were spotted as a negative control. The photographs were taken after a 3 day incubation at 30°C. Right: myc-Mec1p and HA-Tel1p detected by immunoprecipitation followed by Western blot analysis.
(B) Schematic view of the structures of the telomeres (VI-R and XV-L) that were used in the ChIP assay. The arrows indicate the primers used.
(C) ChIP assay of native chromosomal ends. Soluble chromatin of either LSS65 (myc18-MEC1 HA5-TEL1) or a parental strain that lacked the tagged proteins (BY4741) was collected from formaldehyde (w/ crosslink) or mock-treated (w/o crosslink) cells during the exponential growth phase, and immunoprecipitation was carried out with or without the corresponding antibody, followed by multiplex PCR amplification of the telomeric DNA. Two-fold serial dilutions of the input DNA depict the linear range of PCR (lanes 1 to 5).
(D–F) Representative cell cycle ChIP time courses. LSS65 or LSS53 (myc18-MEC1 CDC13-FLAG3) cells were arrested in G1 phase by α factor treatment for 2.5 hr. After washing, synchronous progression through the cell cycle was followed at 20°C. Samples were taken every 30 min and subjected to multiplex ChIP analysis (E). In the Western blot analysis (D), whole-cell extracts were separated in a 5% gel. In the ChIP analysis, immunoprecipitation was performed with or without the corresponding antibodies. Data from (E) are expressed as x-fold enrichment of ACT1 (squares), TEL6 (triangles), and TEL15 (circles) over background levels (F). Error bars represent the standard error from multiple (≥3) independent experiments.
Molecular Cell  , DOI: ( /S (04)00262-X)

3. Figure 2 Mec1p Associates with Telomeres in Late S Phase
(A) Detection of the single-stranded overhang using in-gel hybridization. Cell cycle synchronization experiments were performed as in Figures 1D–1F, and genomic DNA was isolated from cells collected every 30 min. After XhoI digestion, the DNA was separated in a 0.75% agarose gel, and the dried gel was hybridized with a 32P-labeled telomere probe in the native or denatured condition. The incorporation of 32P was quantified using a BAS2500 Phosphorimager (Fuji).
(B and C) Cell cycle analysis of the association of Rfa2p and Rfa3p with the telomeric regions. ChIP analysis was performed as in Figures 1D–1F using LSS120 (myc-MEC1 RFA2-FLAG) or LSS149 (myc-MEC1 RFA3-FLAG). Data are expressed as x-fold enrichment of TEL6 in RPA-IP (solid line) or Mec1p-IP (dashed line) over background levels.
Molecular Cell  , DOI: ( /S (04)00262-X)

4. Figure 3 Reciprocal Association of Tel1p and Mec1p with the Telomeres
(A) Binding of Mec1p to abnormal telomeres. Cells with altered telomere lengths, namely the est2 (telomerase), yku70 (Ku70 protein), and tel1 mutants, were arrested in G1 phase by α factor treatment and subjected to ChIP analysis using the anti-myc antibody. Comparable expression of Mec1p in the mutants was confirmed by Western blot analysis using whole-cell extracts.
(B) Association of Tel1p with telomeres in the Δddc2 mutant. LSS92 (DDC2 Δsml1) and LSS112 (Δddc2 Δsml1) cells were arrested in G1 phase and released for 120 min at 20°C. ChIP analysis was performed using anti-myc and anti-HA antibodies.
(C) Lack of telomere association with Tel1p in the Δyku70 cells. The HA-TEL1 Δyku70 cells arrested in G1 phase were subjected to ChIP analysis using the anti-HA antibody.
(D) Data from (A)–(C) are expressed as x-fold enrichment of TEL6 (white bar) and TEL15 (black bar) over background levels. Error bars represent the standard error from three independent experiments.
Molecular Cell  , DOI: ( /S (04)00262-X)

5. Figure 4
Role of Kinase Activity in the Telomere Associations of Mec1p and Tel1p
(A and B) Effect of kinase-dead mutations on the telomere associations of Mec1p and Tel1p. Cells were arrested in G1 phase and released for 120 min at 20°C. Crosslinked chromatin was subjected to immunoprecipitation with or without the corresponding antibodies.
(C) The cells used in (B) were collected during the exponential growth phase (in the asynchronous condition), followed by the ChIP analysis. The strains used were (A and C) sml1 myc-MEC1 HA-TEL1, sml1 myc-mec1-KD HA-TEL1, sml1 myc-MEC1 Δtel1, and sml1 myc-mec1-KD Δtel1, and (B) myc-MEC1 HA-TEL1 and myc-MEC1 HA-tel1-KD.
(D) Ddc2p-dependent activity of Mec1 kinase against PHAS-I protein and Rad53-SQ4 peptide. Cells (106) were collected during the exponential growth phase and disrupted using glass beads. The anti-myc immunoprecipitates were incubated with [γ-32P]ATP and recombinant PHAS-I (Stratagene) or Rad53-SQ4 peptide at 30°C for 30 min. The reaction products were resolved by SDS-PAGE, and 32P incorporation into the substrates was assessed. Portions of the immunoprecipitates were subjected to Western blot analysis to verify the amounts of Mec1p.
(E and F) The activity of Mec1p peaked in late S phase. myc-MEC1 cells were arrested in the G1 phase for 2.5 hr and released at 20°C. Samples were taken at the indicated time points after release, and Mec1 kinase activity was assayed. The incorporation of 32P into PHAS-I was quantified with a BAS2500 Phosphorimager (Fuji). The relative activity of the Mec1 kinase against PHAS-I is presented as the average ± SD of five independent experiments.
(G) Model of the telomere association of yeast PIKKs. For details, see the text.
Molecular Cell  , DOI: ( /S (04)00262-X)