13 research outputs found
<i>esp1-1</i> functional interaction map derived from the SDL screen.
<p>The 44 genes identified in the <i>esp1-1</i> SDL screen were subjected to Cytoscape analysis. All nodes are significantly enriched (p <0.05) GO Terms in the dataset and coloured nodes represent GO Terms that have been grouped into a significantly enriched category. Grey nodes are GO Terms that were not grouped into an enriched category. Edges define associations between groups and edge thickness indicates the level of significance within the network. Genes identified in the SDL screen that are associated with GO Terms are shown.</p
A Role for the Budding Yeast Separase, Esp1, in Ty1 Element Retrotransposition
<div><p>Separase/Esp1 is a protease required at the onset of anaphase to cleave cohesin and thereby enable sister chromatid separation. Esp1 also promotes release of the Cdc14 phosphatase from the nucleolus to enable mitotic exit. To uncover other potential roles for separase, we performed two complementary genome-wide genetic interaction screens with a strain carrying the budding yeast <i>esp1-1</i> separase mutation. We identified 161 genes that when mutated aggravate <i>esp1-1</i> growth and 44 genes that upon increased dosage are detrimental to <i>esp1-1</i> viability. In addition to the expected cell cycle and sister chromatid segregation genes that were identified, 24% of the genes identified in the <i>esp1-1</i> genetic screens have a role in Ty1 element retrotransposition. Retrotransposons, like retroviruses, replicate through reverse transcription of an mRNA intermediate and the resultant cDNA product is integrated into the genome by a conserved transposon or retrovirus encoded integrase protein. We purified Esp1 from yeast and identified an interaction between Esp1 and Ty1 integrase using mass spectrometry that was subsequently confirmed by co-immunoprecipitation analysis. Ty1 transposon mobility and insertion upstream of the <i>SUF16</i> tRNA gene are both reduced in an <i>esp1-1</i> strain but increased in cohesin mutant strains. Securin/Pds1, which is required for efficient localization of Esp1 to the nucleus, is also required for efficient Ty1 transposition. We propose that Esp1 serves two roles to mediate Ty1 transposition – one to remove cohesin and the second to target Ty1-IN to chromatin.</p></div
All Esp1 domains contribute to Ty1 transposition.
<p>(A,B) The indicated strains, carrying the <i>pGTy1-H3-mhis3AI–URA3</i> plasmid, were patched and induced to undergo transposition on galactose media at the indicated temperatures. After transposition, plates were replica plated to 5-FOA to remove the <i>URA3</i> plasmid as described in the Materials and Methods. Patches are shown after the final step of replica plating. The SC (minimal complete) plates are a control for growth and the colonies present on the SC-HIS plates represent insertion of the <i>Ty1-HIS3</i> element into the genome. (C,D) Triplicate isolates of indicated <i>esp1</i> ts alleles, carrying a <i>pGAL-TyH3mHIS3AI-URA3</i> plasmid (pJBe376) were induced to undergo transposition at semi-restrictive temperature (30°C) by growth in 2% galactose for 24 hours (GAL). One isolate was grown for 24 hours in 2% glucose (D) as a control. <i>SUF16</i> PCR analysis of the extracted yeast genomic DNA is shown and <i>CPR7</i> is a control PCR. All bands in the three lanes for each GAL grown isolate were quantified and summed, then compared to the wild type strain. The relative values of the mutants compared to wild type (value of 1.0) is shown. The amino acid mutations of the <i>esp1</i> alleles have been previously been published: <i>esp1n122</i> (N90S, C511F), <i>esp1b120</i> (K782E, I951T, I1040T), <i>esp1c113</i> (F1327L H1391Y) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005109#pgen.1005109.ref026" target="_blank">26</a>].</p
<i>esp1-1</i> functional interaction map derived from the SL SGA screen.
<p>The 161 genes identified in the <i>esp1-1</i> SL screen were analyzed using Cytoscape. All nodes represent significantly enriched (p <0.05) GO Terms in the dataset. Coloured nodes represent GO Terms that have been grouped into a category (written in the same colour) that is significantly enriched. Edges define associations between groups and edge thickness indicates the level of significance within the network. Genes identified in the SL screen that are associated with GO Terms are shown.</p
<i>esp1</i> and <i>pds1</i> mutants have decreased, whereas cohesin mutant have increased Ty1 mobility.
<p>(A) Transposition frequency of wild type (WT), <i>esp1-1</i>, <i>scc1-73</i>, <i>esp1-1 scc1-73</i>, <i>scc2-4</i>, <i>scc3-1</i>, <i>smc3-1</i>, <i>pds1-128</i>, <i>smc1-259</i>, and <i>spt3Δ</i> (control) cells carrying a <i>CEN</i> plasmid with a marked Ty1element expressed from its endogenous promoter (pBDG922). (B) Transposition frequency of the <i>esp1-1</i> mutant carrying either a vector (pRS315), wild type Esp1 (<i>pESP1</i>), an Esp1 protease domain truncation (<i>pESP1-1112</i>) or an Esp1 catalytic site mutation (<i>pESP1-C1531A</i>) as well as pBDG922. In both (A) and (B), cells were induced to transpose for 4 days at 20°C. A minimum of three independent transformants were quantified. Mutants with transposition frequencies that are significantly different from wild type (A) or pRS315 (B) are shown with one (p<0.05), two (p<0.005) or three asterisks (p<0.0005). (C) Structure of the cohesin ring.</p
The mitotic exit network has a role in Ty1 transposition.
<p>(A) Transposition frequency of wild type (<i>WT</i>), <i>cdc14-1</i>, <i>cdc5-1</i>, <i>cdc15-2</i>, <i>dbf2-1</i>, <i>spo12Δ</i> and <i>slk19Δ</i> cells carrying a plasmid with a marked Ty1element (pBDG922). Mutants with transposition frequencies that are significantly different from WT are shown with three asterisks (p<0.0005). (B) PCR analysis of yeast genomic DNA extracted from indicated strains grown in triplicate for 3 days at 20°C to induce transposition. Upper panel is a <i>SUF16</i> PCR assay whereas the lower panel is a control PCR for the <i>CPR7</i> locus to demonstrate that yeast genomic DNA was present in each sample. Quantification of Ty1 insertion events is shown relative to WT as described in the Materials and Methods.</p
Esp1 physically interacts with Ty1-IN.
<p>(A) Ty1 peptides identified in Esp1-Myc mass spectrometry versus untagged (mock) strain are color coded as follows: Gag/coat protein (blue), PR (yellow), IN (orange) and RT/RNAse H (green). (B) Immunoblot of whole cell lysate (Lysate) and GFP-Trap IP carried out from untagged wild type (No Tag), Esp1-GFP, Scc1-GFP, Pds1-GFP and Ndc80-GFP cells. Expression of a Ty1 element (<i>pGAL1-Ty1-H3</i>) was induced in all strains for 24 hours prior to cell lysis. Blots were probed with anti-GFP and anti-IN (8b11) antibodies. (C) Immunoblot of whole cell lysate (Lysate) and GFP-Trap IP carried out from Esp1-Myc or untagged wild type (No Tag) cells carrying a pGAL-GFP-LacZ-Ty1-IN plasmid (Ty1-IN) or pGAL-GFP-lacZ (vector) control. Cells were either grown in glucose (D) or galactose (GAL) for 24 hours to repress or induce GFP-LacZ-Ty1-IN expression, respectively. The asterisk marks a background band that is present in the lysate of the cells grown in glucose but is not detected in the GFP-Trap IP.</p
Ty1 cDNA levels are not affected in <i>esp1</i> and <i>pds1</i> mutants.
<p>(A) Southern blot analysis of <i>AflII</i> digested yeast genomic DNA isolated from the indicated mutants carrying the <i>pGTy1-H3-mhis3AI</i> plasmid after 24 hour induction with 2% galactose. One wild type (WT) sample was also grown in 2% glucose (GLU) as a control for <i>TY1</i> element expression. The blot was probed with a radiolabeled <i>HIS3</i> gene. Shown are the fragment sizes for Ty1 cDNA (∼2.4kb), the endogenous <i>HIS3</i> locus of the S288C deletion strain with 200bp deleted (<i>his3Δ1</i>, ∼6.5kb) and the <i>pGTy1-H3-mhis3AI</i> plasmid which is linearized (∼14kb). Below each lane is the quantitative ratio of Ty1 cDNA signal to <i>his3Δ1</i> signal. (B) Southern blot analysis of <i>PvuII</i> digested yeast genomic DNA isolated from the indicated mutants after 2 days growth at 20°C. Endogenous Ty1 elements and cDNA were detected with a radiolabelled <i>PvuII/SnaBI</i> Ty1 element fragment. Below each lane is the quantitative ratio of the Ty1 endogenous cDNA (∼2kb) to an endogenous Ty element (Ty1 control). <i>spt3Δ</i> serves as a negative control for Ty1 cDNA levels. (C) Endogenous Ty1-Gag processing in wild type (WT) versus <i>esp1-1</i> cells grown at 25°C or incubated for 6 hours at 30°C assessed by immunoblot. Blot was probed with anti-Gag antibody. p49 = unprocessed Gag; p45 = processed Gag. <i>spt3Δ</i> serves as a negative control for Gag expression.</p
Esp1 interacts with Ty1-IN in G1 and G2/M phase cells.
<p>Untagged (No Tag) and Esp1-GFP tagged cells carrying the <i>pGAL1-Ty1-H3</i> element were induced for 24 hours with 2% galactose, then arrested in G1 phase with mating pheromone (α-factor), S phase with hydroxyurea (HU) and G2/M phase with Nocodazole (Nz). Immunoblot of whole cell lysate (Lysate) and GFP-Trap IP from Log, α-factor, HU and Nz arrested cells is shown. Blots were probed with anti-GFP and anti-IN (8b11) antibodies.</p
Genes identified in Esp1-13Myc mass spectrometry screen implicated in Ty1 transposition.
<p><sup>a</sup>All mutants have decreased Ty1 transposition except for <i>YML105C</i> and <i>YOL159C</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005109#pgen.1005109.ref040" target="_blank">40</a>]</p><p><sup>b</sup>Mutants that suppress the hypertransposition phenotype of <i>rtt101Δ</i> and <i>med1Δ</i> mutants [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005109#pgen.1005109.ref045" target="_blank">45</a>]</p><p><sup>c</sup><i>rtt</i> mutants have increased levels of Ty1 transposition [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005109#pgen.1005109.ref043" target="_blank">43</a>]</p><p><sup>d</sup><i>SPT16</i> is a high-copy suppressor of Ty1-δ insertion mutations [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005109#pgen.1005109.ref050" target="_blank">50</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005109#pgen.1005109.ref051" target="_blank">51</a>]</p><p><sup>e</sup><i>spt</i> mutants suppress <i>his4-912δ</i> Ty insertion mutations [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005109#pgen.1005109.ref052" target="_blank">52</a>]</p><p>Genes identified in Esp1-13Myc mass spectrometry screen implicated in Ty1 transposition.</p