1. Elucidation of the DNA End-Replication Problem in Saccharomyces cerevisiae 
Julien Soudet, Pascale Jolivet, Maria Teresa Teixeira 
Molecular Cell 
Volume 53, Issue 6, Pages (March 2014)
DOI: /j.molcel
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2. Molecular Cell 2014 53, 954-964DOI: (10.1016/j.molcel.2014.02.030)
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3. Figure 1
The Length of the Telomeric G-Strand Containing the 3′ Overhang Is Stable over Several Cell Divisions
(A) Cells were synchronized in G1 phase (+αF, 0 population doublings [PDs]) and then released for one cell cycle in the presence of BrdU (1 PD). Then the cells were washed and grown for an additional 4 PDs (5 PDs). In this scheme, the strands that have incorporated the BrdU (black circles) are diluted over the generations. Because the telomeric sequence in S. cerevisiae consists of TG1-3/C1-3A repeats, BrdU is incorporated exclusively into the newly synthesized G-strand of the leading telomere. Throughout the experiment, TetO2-TLC1 cells were kept in doxycyline (+DOX) to repress telomerase. See Figures S1A and S1B for further details.
(B) Quantification of BrdU incorporation. Identical quantities of DNA from samples 0, 1, and 5 PDs were spotted on a membrane. BrdU was detected using a monoclonal antibody (upper panel). The mean quantity of BrdU left in DNA from three independent experiments is indicated (±SEM) considering 0 PD as 0% and 1 PD as 100%. The same samples were hybridized with a CA probe as a loading control (lower panel). DNA content analysis by fluorocytometry for the three time points is shown. The effects of BrdU on cell viability were also checked (see Figures S1C and S1D).
(C) DNA from the above samples was extracted, denatured (input fraction), and submitted to BrdU immunoprecipitation with α-BrdU antibody (IP fraction). During telomere-PCR, terminal transferase adds dCTP to the 3′ ends. Subsequent PCR using primers targeting both the Y’ subtelomeric elements conserved at several telomeres and the C-tail selectively amplify the 3′-containing strands (see also Figures S1E and S1F).
(D) Representative telomere-PCR products of input and IP fractions of the indicated time points. Pink bars correspond to the mean length of the smears.
(E) Quantification of the shortening rate of Y’ telomeres between 1 PD and 5 PDs in base pairs (bp) per PD (n = 3 independent experiments, error bars correspond to SEM).
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2 Using the TPX Assay to Determine G-Tail Length
(A) Rationale of TPX. DNA treated or not treated with Exo I is subjected to telomere-PCR. After separation by electrophoresis, the difference in length between the products is considered as the G-tail length. See also Figure S2 for in vitro controls.
(B) TPX on WT, tel1Δ, and ku80Δ strains. Telomere-PCR products targeting the telomere 6R were electrophoresed, and the mean length of the telomeric smear was determined (pink bars).
(C) Estimation of 6R overhang length by TPX for WT, tel1Δ, and ku80Δ strains (n = 4 independent experiments, error bars correspond to SEM). nt: nucleotides.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3 Dynamics of the Telomeric G-Tail during the Cell Cycle
(A and B) Telomerase-repressed TetO2-TLC1 (+DOX) or TetO2-TLC1 tel1Δ cells were synchronized with α factor and released into the cell cycle at 24°C (A). Cell samples were collected every 15 min and (B) DNA content was measured by fluorocytometry.
(C and D) TPX products targeting the 6R telomeres were subjected to gel electrophoresis and smear profiles were analyzed. The mean length of the telomeric tract at point 0 is indicated on the right side of each panel.
(E) Quantification by TPX of the 3′ overhang lengths (n = 3 independent experiments, error bars correspond to SEM). Refer to Figure S3 for results for the Y’ telomeres.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
Analysis of the G-Tail Dynamics at the Leading and Lagging Telomeric Strands over the Cell Cycle
(A) Cells were synchronized in G1 phase (+αF) and then released for the first cell cycle in the presence of BrdU at 24°C. α factor was added again at 60 min to synchronize the cells in the next G1 phase. Then cells in the second G1 phase were washed from the BrdU and grown at 24°C during a second cell cycle. In this experimental scheme, BrdU (black circles) is first incorporated into the newly synthesized leading strand (Lead); then, in the second cell cycle, it is contained in the strand that serves as the template for lagging strand (Lagg) synthesis.
(B) DNA content analysis by fluorocytometry for G1 phase (0 min) and S phase (45 min) for the two consecutive cell cycles for the TetO2-TLC1 (+DOX) and TetO2-TLC1 tel1Δ cells.
(C) DNA was extracted in G1 phase (0 min) and S phase (45 min) of the two consecutive cell cycles for the TetO2-TLC1 (+DOX) cells, treated or not treated with Exo I, BrdU immunoprecipitated, and submitted to telomere-PCR targeting the 6R telomeres.
(D) The same as in (C) using the TetO2-TLC1 tel1Δ strain.
(E) Quantification of the 3′ overhang lengths at newly synthesized leading and lagging telomeric strands in G1 and S phases (n = 3 independent experiments, error bars correspond to SEM). N/A: not applicable. Control for BrdU effects on TPX assay is shown in Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
Cdc13 Couples the Replication of Leading and Lagging Telomeric Strands
(A) Telomerase-depleted cells (+DOX) were synchronized in G1 phase (+αF) and released in the cell cycle at 18°C in a BrdU-containing medium. At the time of replication, BrdU (black circles) is incorporated into the G-strand of newly synthesized leading telomeres (Lead, red) and in the subtelomeric C-strand of newly synthesized lagging telomeres (Lagg, green). Then DNA is extracted at different points of the cell cycle and immunoprecipitated with α-BrdU antibodies. The different telomeric strands are subsequently detected by a CA- or a TG-probe hybridized to the newly synthesized leading or lagging telomere, respectively.
(B) Cell samples were collected at different time points for the indicated strains, and DNA content was measured by fluorocytometry.
(C) Quantification of strand replication during the cell cycle in the indicated strain. Quantifications were performed on dot blots shown in Figure S5A considering the IP at t = 0 as 0% of replication and the last time point as 100% of replication and fitted to a three-parameter sigmoid. One representative clone out of three independent experiments is shown (see Figures S5B and S5C for the other two clones and Figures S5D–S5I for other conditions).
(D) Same as in (C) using a strain containing the cdc13-5 allele. Refer to Figure S5J for the dot blots and Figure S5K and S5L for the other two clones.
(E) Differences in time between lagging- and leading-strand replication at 50% of replication (t % of replication of lagging − t % of replication leading) (n = 3 independent experiments, error bars correspond to SEM).
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
Revised Model of the DNA End-Replication Problem in S. cerevisiae
When the replisome reaches the telomere, Cdc13 ensures the timely replication of the lagging telomere (b). The last Okazaki fragment is initiated near the 3′ end, resulting in a gap of 5–10 nt (a). Synthesis of the leading telomere may proceed up the 5′ end of the template, generating a blunt intermediate (c), which is rapidly processed through a Tel1-dependent pathway generating a 3′ overhang of about 40 nt (d). Then the C-strand fill in occurs, placing again a last Okazaki fragment at 5–10 nt from the end (ea). In this scheme, telomeres shorten at a rate of 2.5–5 bp/PD (a).
Molecular Cell  , DOI: ( /j.molcel )
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