Chromosome truncation at internal TG repeats.

<p>(A) Schematic of the 81 bp TG (TG₈₁) or 250 bp TG (TG₂₅₀) repeat at the <i>ADH4</i> locus on chromosome VII-L. The <i>ADH4</i> locus was replaced with a DNA fragment containing the <i>KanMX</i> gene and the <i>URA3</i>^<i>Kl</i> gene. The TG₈₁ or TG₂₅₀ sequence was introduced between <i>KanMX</i> and <i>URA3</i>^<i>Kl</i>. The cassette with no TG sequence was called as TG₀. The centromere is shown as a circle on the left (CEN) and the telomere is shown as repetitive arrows on the right. The black line indicates a hybridization probe for Southern blot (Fig 1C). TG-telomere cells contain a telomere at the position where the TG₂₅₀ repeat is inserted (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005283#pgen.1005283.s001" target="_blank">S1 Fig</a> for more details). (B) Schematic of experimental protocol to detect <i>URA3</i>^<i>Kl</i> marker loss after telomere addition at the TG repeat sequences. (C) Effect of TG₈₁ or TG₂₅₀ insertion on <i>URA3</i> marker loss. Cells containing the TG₀, TG₈₁ or TG₂₅₀ cassette were first maintained in medium selective for <i>URA3</i> and then transferred to non-selective medium. Saturated cultures were diluted and spread out on 5-FOA plates to determine the rate of <i>URA3</i>^<i>Kl</i> marker loss. <i>URA3</i>^<i>Kl</i> marker loss rate per generation was quantified through fluctuation analysis. Error bars represent 95% confidence intervals. Number in parentheses indicates rate relative to cells containing the TG₀ cassette. Rap1 contains a BRCT domain, two Myb domains, a transcription activation (TA) domain and a C-terminal RCT domain. The <i>rap1-ΔN</i> and the <i>rap1-ΔC</i> mutation lack the BRCT and the RCT domain, respectively. (D) Effect of TG₂₅₀ insertion on chromosome truncation. Cells containing the TG₀ or TG₂₅₀ cassette were first maintained in medium selective for <i>URA3</i> and then diluted 1000-fold in non-selective medium to allow cells to grow for one day (10 generations) and aliquots were collected for genomic DNA preparation. This cycle was repeated again. DNA was digested with HindIII and analyzed by Southern blot using the probe shown in A. DNA breakage at TG₂₅₀ generates a 0.7 kb fragment (marked with CUT) from the 6.9 kb fragment (PRE). The probe also detects a 1.8 kb HindIII fragment (Control) from the <i>SMC2</i> locus on chromosome VI. TG-telomere (TG-tel) serves as a control to detect DSB induction at the TG₂₅₀ sequence. (E) Effect of TG₂₅₀ insertion on replication fork pausing. Cells containing the TG₀ or TG₂₅₀ cassette were first maintained in medium selective for <i>URA3</i> and then transferred to non-selective medium to allow cells to undergo cell division for 4 hr. CsCl gradient purified DNA was digested with AflII and XbaI and analyzed by two-dimensional gel electrophoresis using the indicated probe. The probe detects a 6.3 kb AflII-XbaI fragment. The TG₂₅₀ repeat locates 3.0 kb from the AflII site and 2.9 kb from the XbaI site. RFP represents replication fork pausing. The arrow indicates the direction of replication fork movement. There is a highly active replication origin 40 kb proximal to the repeat insert site (the <i>ADH4</i> locus) on chromosome VII [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005283#pgen.1005283.ref082" target="_blank">82</a>].</p

DSB induction at the LacO₁₆ repeat after LacI-Rap1 expression.

<p>(A) Schematic of the LacO₁₆ repeat at the <i>ADH4</i> locus on chromosome VII-L. The <i>ADH4</i> locus was marked with a DNA fragment containing the <i>KanMX</i> gene and the <i>URA3</i>^<i>Kl</i> gene. The LacO₁₆ sequence is inserted between the BamHI and EcoRI site. Black bar indicates a hybridization probe for Southern blot. The forward primer (F) and reverse primer (R) were used for a TdT-based DNA end detection assay. Primer F and R are 0.52 kb and 1.1 kb away from the LacO₁₆ insertion site, respectively. (B) Detection of DNA breakage by Southern blot. Wild-type or <i>rad52Δ yku70Δ</i> mutant cells containing the LacO₁₆-URA3 cassette were transformed with pGAL-LacI-RAP1 or pGAL-LacI. Transformants were initially grown in 2% sucrose and then incubated with 2% galactose and 0.5% glucose for 4 hr. Genomic DNA was digested with NcoI and then analyzed by Southern blot using the <i>KanMX</i> gene and a fragment from chromosome I as a probe. Hybridization detects two NcoI-fragments; one from chromosome I (5 kb, marked with INT) and the other from the chromosome VII region (11.6 kb, marked with PRE). DNA breakage at LacO₁₆ generates a 1.3–1.7 kb fragment (marked with CUT) from the 11.6 kb fragment. (C) Detection of DNA ends by TdT-dependent dCTP addition. LacO₁₆-URA3 cells carrying pGAL-LacI-RAP1 or pGAL-LacI were treated as in (B). Genomic DNA was incubated with TdT using dCTP as a substrate and then subjected to PCR using either the forward primer or the reverse primer with a poly(dG)-oligonucleotide. As control, the <i>SMC2</i> locus was amplified by PCR.</p

DSB induction at an artificially elongated telomere.

<p>(A) Schematic of modified VII-L telomeres. Telomeres marked the <i>KanMX</i> marker are generated near the <i>ADH4</i> locus. Triangles and black bars represent TG and lacO sequence, respectively. The VII-L subtelomere sequence is deleted. The black line indicates a hybridization probe. Each chromosome end contains a wild-type length telomere (~300 bp) before LacI-Rap1 expression. Drawing not to scale. (B) Schematic of experimental protocol to detect DNA breaks at an extended telomere. LacI-Rap1 expression converts TG₃₃-LacO₁₆-telomeres to a long telomere mimic. Telomeres become shortened 3–5 bp per generation because of the end-replication problem. Cdc13 binds single-stranded telomeric TG DNA and blocks DNA degradation. Telomere shortening or DSB induction needs to trim off ~300 bp telomeric TG sequence to initiate degradation of the LacO₁₆ repeat. Once the LacO₁₆ repeat is degraded, telomere addition occurs at the TG₃₃ repeat, generating TG-telomere. (C) Effect of LacI-Rap1 expression on the length of LacO₄-telomeres. Cells containing the LacO₄-telomere were transformed with pGAL-LacI-RAP1 or pGAL-LacI. Transformed cells were grown in sucrose to full growth. The culture was then diluted 1000-fold in 2% galctose and 0.5% glucose to allow cells to undergo cell division for 24 hr and aliquots were collected for genomic DNA preparation. This cycle was repeated three times. DNA was digested with HindIII and analyzed by Southern blot using the probe shown in (A). The probe detects the LacO₄-telomere and a 1.8 kb HindIII fragment (Control) from the <i>SMC2</i> locus on chromosome VI. (D) Effect of LacI-Rap1 expression on the length of TG₃₃-LacO₁₆-telomeres. Cells containing the TG₃₃-LacO₁₆-telomere (TG₃₃-LacO₁₆-tel cells) were transformed with pGAL-LacI-RAP1 or pGAL-LacI. Transformants were analyzed as in (C). As control, DNA from cells containing TG-telomere was examined together. The band labeled PRE indicates a fragment containing TG₃₃-LacO₁₆ telomere (~1.2 kb). After DSB induction within or near the LacO₁₆ repeat, this band is converted to a new band (~0.8 kb), which is similar to the fragment containing wild-type length TG-telomeres. The probe also detects a 1.8 kb HindIII fragment (Control) from the <i>SMC2</i> locus on chromosome VI. (E) Effect of telomerase loss on the length of TG₃₃-LacO₁₆-telomeres. TG₃₃-LacO₁₆-tel cells were transformed with pGAL-LacI-RAP1 whereas TG₃₃-LacO₁₆-tel <i>est1Δ</i> cells carrying the <i>URA3</i>-marked <i>EST1</i> plasmid were transformed with pGAL-LacI. Transformants were streaked on plates containing 5-FOA and colonies of Ura^- cells were inoculated in 2% galactose and 0.5% glucose medium and grown to the late log phase (1st dilution). The culture was diluted 1000-fold to allow cells to undergo cell division for 24 hr. This cycle was repeated twice. DNA was analyzed as in (D). TG₃₃-LacO₁₆-tel cells in sucrose (0 dilution) or <i>est1Δ</i> mutants carrying the <i>URA3</i>-marked <i>EST1</i> plasmid (E) in sucrose were examined as control. (F) Effect of r<i>ad52Δ</i> mutation on the length of TG₃₃-LacO₁₆-telomeres after LacI-Rap1 expression. TG₃₃-LacO₁₆-tel wild-type or <i>rad52Δ</i> cells were transformed with pGAL-LacI-RAP1. Transformed cells were analyzed as in (D).</p

DNA replication fork pausing at the LacO₁₆ repeat after LacI-Rap1 expression.

<p>(A) LacO₁₆-URA3 cells carrying pGAL-LacI-RAP1, pGAL-LacI or the control vector were cultured as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005283#pgen.1005283.g004" target="_blank">Fig 4B</a>. CsCl gradient purified DNA was digested with AflII and XbaI and analyzed by two-dimensional gel electrophoresis using the indicated probe. The probe detects a 6.3 kb AflII-XbaI fragment. The LacO₁₆ repeat locates 3.0 kb from the AflII site and 2.9 kb from the XbaI site. RFP represents replication fork pausing. Note that some parts of RFP signal are smearing (ST). The number (%) below each panel denotes the ratio of the signal of RFP to that of total replication intermediates. The arrow indicates the direction of replication fork movement. There is a highly active replication origin 40 kb proximal to the LacO₁₆ repeat insert site (the <i>ADH4</i> locus) on chromosome VII [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005283#pgen.1005283.ref082" target="_blank">82</a>]. (B) Effect of <i>mec1Δ</i> mutation on DNA breakage induction. LacO₁₆-URA3 <i>MEC1</i> or <i>mec1Δ</i> cells were transformed with pGAL-LacI (I) or pGAL-LacI-RAP1 (R). Transformants were cultured and DNA was analyzed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005283#pgen.1005283.g004" target="_blank">Fig 4B</a>. Cells contain an <i>sml1Δ</i> mutation. Hybridization detects two NcoI-fragments; one from chromosome I (5 kb, marked with INT) and the other from the chromosome VII region (11.6 kb, marked with PRE). DNA breakage at LacO₁₆ generates a 1.3–1.7 kb fragment (marked with CUT) from the 11.6 kb fragment. (C) Effect of <i>mrc1Δ</i> or <i>tof1Δ</i> mutation on DNA breakage induction. LacO₁₆-URA3, LacO₁₆-URA3 <i>mrc1Δ</i> or LacO₁₆-URA3 <i>tof1Δ</i> cells were transformed with pGAL-LacI or pGAL-LacI-RAP1. Transformants were cultured and DNA was analyzed as in (B).</p

Effect of Rap1 tethering on homologous recombination between the <i>ura3-ΔN</i>^<i>Kl</i> gene and the <i>ura3-ΔC</i>^<i>Kl</i> gene.

<p>(A) Schematic of the ura3-ΔC-LacO cassette on chromosome VII-L and the ura3-ΔN cassette on chromosome V-R. The <i>YER186</i> locus on chromosome V was replaced with the ura3-ΔN cassette marked with the <i>HphMX</i> gene. The <i>ADH4</i> locus on chromosome VII was replaced with the <i>KanMX</i>-marked cassettes containing the <i>ura3-ΔC</i>^<i>Kl</i> truncated gene and different copies (n) of the lacO sequence. There is no essential gene from the <i>ADH4</i> or <i>YER186</i> locus to the corresponding chromosome end. (B) Effect of LacI-Rap1 expression on homologous recombination near the LacO₀, LacO₄, LacO₈ or LacO₁₆ cassette. Fusion proteins contain the DNA-binding domain of LacI (black square). This LacI construct lacks the C-terminal oligomerization domain but contains the nuclear localization signal (PKKKRKV) derived from the SV40 Large T-antigen. Cells carrying pGAL-LacI-RAP1 or the control vector were grown in 2% sucrose and then transferred to 2% galactose and 0.5% glucose. Saturated cultures were diluted and spread on uracil dropout plates. <i>URA3</i>^<i>Kl</i> homologous recombination rate per generation was determined through fluctuation analysis. Error bars represent 95% confidence intervals. Number in parentheses indicates rate relative to LacO₀ cells carrying the control vector. (C) Effect of LacI, LacI-GAL4 or LacI-Rap1 (224–663) expression on homologous recombination near the LacO₁₆ cassette. Cells carrying pGAL-LacI, pGAL-LacI-RAP1, pGAL-LacI**-RAP1, pGAL-LacI-GAL4, pGAL-LacI-RAP1 (224–663) or the control vector were cultured and examined as in (B). Number in parentheses indicates rate relative to cells containing the LacO₁₆ cassette and carrying the control vector. The construction of LacI-Rap1 (224–663) is shown in (B). (D) Effect of <i>rif1Δ rif2Δ</i> or <i>sir3Δ sir4Δ</i> mutation on homologous recombination near the LacO₁₆ cassette. Wild-type, <i>rif1Δ rif2Δ</i> or <i>sir3Δ sir4Δ</i> mutant cells carrying pGAL-LacI-RAP1 were cultured and examined as in (C). Number in parentheses indicates rate relative to wild-type cells containing the LacO₁₆ cassette.</p

Requirement of Rap1 on chromosome truncation at TG repeats.

<p>(A) Effect of <i>rap1</i>-degron mutation on colony formation in the presence of CuSO₄. Wild-type or <i>rap1</i>-degron mutant (<i>rap1-(Δ)</i>) cells were serially diluted (10-fold) and spotted on medium containing 0, 0.05 or 0.5 mM CuSO₄. (B) Effect of CuSO₄ concentration on Rap1-(Δ) protein expression. Wild-type or <i>rap1-(Δ)</i> cells were initially grown in the absence of CuSO₄ and then incubated with the indicated concentrations of CuSO₄ for 6 hr. Aliquots of cells were collected and subjected to immunoblotting analysis with anti-Rap1 antibodies. (C) Effect of Rap1 depletion on <i>URA3</i>^<i>Kl</i> marker loss. Wild-type or <i>rap1-(Δ)</i> cells containing the TG₂₅₀ cassette were first maintained in medium selective for <i>URA3</i> and then transferred to non-selective medium containing 0.05 mM CuSO₄. Saturated cultures were diluted and spread on 5-FOA plates to determine the rate of <i>URA3</i>^<i>Kl</i> marker loss. <i>URA3</i>^<i>Kl</i> maker loss was determined as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005283#pgen.1005283.g001" target="_blank">Fig 1C</a>.</p

The sequences of the primers used in the amplification of corresponding genes.

<p>The sequences of the primers used in the amplification of corresponding genes.</p

Co-localizations of ADA2A, ADA2B, GCN5 with AATF protein in HEK293 cells.

<p>Intracellular co-localization of CFP-conjugated ADA2A (A), ADA2B (B), GCN5 (C), empty vector expressing CFP alone (D) and YFP-conjugated AATF proteins (middle column). Yellow color in the merged image (last column) indicates co-localization. Live cell images were captured by confocal microscopy and pseudo-coloured red (CFP) and green (YFP). Insets show single transfections. Upper right corners of nuclei are marked with a curved line. The last column shows merged images. Scale bar is 5 μm.</p

Effect of co-expression of ADA2A, ADA2B, GCN5 with AATF on the expression of an MDM2 promoter-luciferase reporter gene.

<p>U2OS cells were transfected with a reporter plasmid encoding a luciferase gene under the control of the MDM2 promoter or co-transfected with the reporter plasmid and plasmids expressing ADA2A, ADA2B, GCN5 with AATF protein partner as indicated. Luciferase activity was measured 48 h post-transfection. Results represent the average ± SD from three independent assays. Co-expression of AATF with ADA2A, ADA2B or GCN5, significantly reduced the activation imparted by AATF on MDM2 promoter-directed transcription (<i>p</i> <0.01).</p