11 research outputs found
Analysis of chromosomal rearrangements after replication restart
Impediments to DNA replication are known to induce gross chromosomal rearrangements (GCRs) and copy-number variations (CNVs). GCRs and CNVs underlie human genomic disorders and are a feature of cancer. During cancer development, environmental factors and oncogene-driven proliferation promote replication stress. Resulting GCRs and CNVs are proposed to contribute to cancer development and therapy resistance.
Using an inducible system that arrests replication forks at a specific locus in fission yeast, chromosomal rearrangement was investigated. In this system, replication restart requires homologous recombination. However, it occurs at the expense of gross chromosomal rearrangements that occur by either faulty template usage at restart or after the correctly restarted fork U-turns at inverted repeats. Both these mechanisms of chromosomal rearrangement generate acentric and reciprocal dicentric chromosomes. The work in this thesis analyses the timing of replication restart and appearance of chromosomal rearrangements in a single cell cycle after induction of fork stalling. This research also identifies the recombination-dependent intermediates corresponding to the two pathways of rearrangements. Moreover, the DNA integrity checkpoint responses after replication fork arrest, homologous recombination dependent replication restart, and the accumulation of GCRs are investigated
Optimisation of the Schizosaccharomyces pombe urg1 expression system
The ability to study protein function in vivo often relies on systems that regulate the presence and absence of the protein of interest. Two limitations for previously described transcriptional control systems that are used to regulate protein expression in fission yeast are: the time taken for inducing conditions to initiate transcription and the ability to achieve very low basal transcription in the "OFF-state". In previous work, we described a Cre recombination-mediated system that allows the rapid and efficient regulation of any gene of interest by the urg1 promoter, which has a dynamic range of approximately 75-fold and which is induced within 30-60 minutes of uracil addition. In this report we describe easy-to-use and versatile modules that can be exploited to significantly tune down P urg1 "OFF-levels" while maintaining an equivalent dynamic range. We also provide plasmids and tools for combining P urg1 transcriptional control with the auxin degron tag to help maintain a null-like phenotype. We demonstrate the utility of this system by improved regulation of HO-dependent site-specific DSB formation, by the regulation Rtf1-dependent replication fork arrest and by controlling Rhp18(Rad18)-dependent post replication repair
Polymerase Ξ΄ replicates both strands after homologous recombination-dependent fork restart
To maintain genetic stability DNA must be replicated only once and replication completed even when individual replication forks are inactivated. Because fork inactivation is common, the passive convergence of an adjacent fork is insufficient to rescue all inactive forks. Thus, eukaryotic cells have evolved homologous recombination-dependent mechanisms to restart persistent inactive forks. Completing DNA synthesis via Homologous Recombination Restarted Replication (HoRReR) ensures cell survival, but at a cost. One such cost is increased mutagenesis caused by HoRReR being more error prone than canonical replication. This increased error rate implies that the HoRReR mechanism is distinct from that of a canonical fork. Here we exploit the fission yeast Schizosaccharomyces pombe to demonstrate that a DNA sequence duplicated by HoRReR during S phase is replicated semi-conservatively, but that both the leading and lagging strands are synthesised by DNA polymerase delta
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Analysis of chromosomal rearrangements after replication restart
Impediments to DNA replication are known to induce gross chromosomal rearrangements (GCRs) and copy-number variations (CNVs). GCRs and CNVs underlie human genomic disorders and are a feature of cancer. During cancer development, environmental factors and oncogene-driven proliferation promote replication stress. Resulting GCRs and CNVs are proposed to contribute to cancer development and therapy resistance. Using an inducible system that arrests replication forks at a specific locus in fission yeast, chromosomal rearrangement was investigated. In this system, replication restart requires homologous recombination. However, it occurs at the expense of gross chromosomal rearrangements that occur by either faulty template usage at restart or after the correctly restarted fork U-turns at inverted repeats. Both these mechanisms of chromosomal rearrangement generate acentric and reciprocal dicentric chromosomes. The work in this thesis analyses the timing of replication restart and appearance of chromosomal rearrangements in a single cell cycle after induction of fork stalling. This research also identifies the recombination-dependent intermediates corresponding to the two pathways of rearrangements. Moreover, the DNA integrity checkpoint responses after replication fork arrest, homologous recombination dependent replication restart, and the accumulation of GCRs are investigated
Determinant of Selective Removal (DSR) sequences can reduce protein levels expressed from P<i><sub>urg1lox</sub></i>.
<p>(<b>A</b>). Schematic of the P<i><sub>urg1lox</sub></i> locus following RCME to introduce yEGFP under the control of the <i>urg1</i> promoter. The resulting transcript encodes the ORF followed immediately after the stop codon by one of a variety of DSR sequences. These act to target the transcript to the nuclear exosome. (<b>B</b>). The strains created with the yEGFP ORF with and without DSR sequences present in the 3β UTR: AW640 (NO DSR), AW702 (8XmDSR), AW726 (1XDSR), AW728 (2XDSR), AW730 (3XDSR), AW732 (4XDSR), AW694 (5XDSR), AW696 (6XDSR), AW698 (7XDSR), AW700 (8XDSR) and AW638 (<i>spo5</i>DSR) (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083800#pone-0083800-t001" target="_blank">Table 1</a>). The strains were cultured in EMM+L to βΌ5Γ10<sup>6</sup> cells at 30Β°C (uracil absent - P<i><sub>urg1lox</sub></i> OFF). Uracil was added at 0.25 mg/ml and cells grown for 120 minutes (P<i><sub>urag1lox</sub></i> ON). Total protein extracts from un-induced P<i><sub>urg1lox</sub></i> OFF cells (lanes 2β12) and induced P<i><sub>urg1lox</sub></i> ON cells (lanes 13β23) were separated by SDS PAGE prior to Western blotting using anti-GFP to detect yEGFP (upper panels) and anti-tubulin for a loading control (lower panel). Lane 1 β=β WT control <i>urg1</i><sup>+</sup> strain (AW501). (<b>C</b>) Comparison of the induced P<i><sub>urg1lox</sub></i>-yEGFP levels in B. Induced cell total protein extracts were used to estimate the fold-decrease in protein levels after induction. The NO DSR sample (lane 13 panel B) was serially diluted using SDS sample buffer and analysed (lanes 2 to 7) alongside undiluted induced 3XDSR (AW730 - lane 8), 4XDSR (AW732 - lane 9), 6XDSR (AW696 - lane 10), 8XDSR (AW700 - lane 11) and <i>spo5</i>DSR (AW638 - lane 12) samples. Lane 13 β=β undiluted un-induced NO DSR sample (the same as lane 2 panel B). (<b>D</b>) The kinetics of yEGFP accumulation is unaffected when using a DSR element. Time-course showing yEGFP protein levels in NO DSR (AW640 - lanes 2 to 8) and <i>spo5</i>DSR (AW638 - lanes 10 to 16) cells after addition of uracil at 0.25 mg/ml to induce P<i><sub>urg1lox</sub></i>. Analysis of yEGFP levels by western blot as described in B. Samples taken at time-points shown (mins). Lanes 1 and 9 β=β control <i>urg1</i><sup>+</sup> strain (AW501). (<b>E</b>) Over-expression of mRNAs containing DSR sequences does not affect cells growth/viability. Strains shown were serially diluted 10-fold in water and spotted on EMM+L plates supplemented with uracil at concentration shown. Pictures were taken after 3 days at 30Β°C.</p
Use of an auxin-inducible degron allows for the generation of a conditional <i>rhp18</i> mutant strain.
<p>(<b>A</b>) Cartoon of the IAA17 degron system: addition of auxin allows binding of the TIR adaptor (fused to Skp1) to the IAA17 tag (IAA), which is fused to the target protein. This induces ubquitination and proteasome degradation. (<b>B</b>) Strains AW617 (<i>rhp18</i><sup>+</sup>), YDP273 (<i>rhp18</i>Ξ) and YDP231 (<i>rhp18</i>Ξ, <i>P<sub>urg1lox</sub>-rhp18-HAIAA17-spo5DSR</i>) were serially diluted 10-fold in water and spotted on EMM+L plates supplemented as shown with uracil at 0.25 mg/ml, NAA at 0.5 mM and/or 4NQO at 400 nM. Time of incubation at 30Β°C: Top panels 3 days, bottom panels 5 days. (<b>C</b>) Schematic of experimental procedure used in D. HU β=β hydroxyurea, NAA β=β 1-naphthaleneacetic acid. (<b>D</b>) Ubiquitination of PCNA is abolished in <i>P<sub>urg1lox</sub>-rhp18-HAIAA7-spo5DSR</i> cells in the presence of NAA. Logarithmically growing AW617 (<i>rhp18</i><sup>+</sup>), YDP273 (<i>rhp18-</i>delete), YDP210 (<i>rhp18-</i>delete, <i>P<sub>urg1lox</sub>-rhp18-HAIAA17</i>) and YDP231 (<i>rhp18-</i>delete, <i>P<sub>urg1lox</sub>-rhp18-HAIAA17-spo5DSR</i>) cells cultured in EMM+L at 30Β°C untreated (β) or treated with 10 mM HU (+) and grown for a 120 minutes or grown for 120 minutes in the presence of NAA at 0.5 mM or uracil at 0.25 mg/ml. Total protein extracts were separated by SDS PAGE prior to Western blotting using anti-PCNA antibody. (<b>E</b>). The auxin degron promotes protein degradation upon βshut-offβ. P<i><sub>urg1/lox</sub></i>. YDP210 (<i>rhp18-</i>delete, <i>P<sub>urg1lox</sub>-rhp18-HAIAA17</i>) cells were grown in EMM+L and <i>P<sub>urg1lox</sub></i> induced by the addition of uracil at 0.25 mg/ml. After 3 h induction, cells were pelleted by centrifugation, washed twice in EMM+L and re-suspended in EMM+L. Samples taken at time-points shown (mins). Total protein extracts were separated by SDS PAGE prior to Western blotting revealed protein levels using anti-HA to detect Rhp18-HAIAA17 (upper panels) and anti-tubulin to detect tubulin as a loading control (lower panel). WT represents control strain AW501 (<i>h</i><sup>β</sup>, <i>leu1-32</i>).</p
Use of the <i>S. pombe spo5</i> gene DSR element allows for tighter regulation of Rtf1 expression in an RTS1-dependent replication fork stall system.
<p>(A) Schematic illustration of inverted <i>ura4</i> repeat double RTS1 (<i>RuiuR</i>) construct. <i>RTS1</i> is a polar replication fork barrier. The triangular indent indicates the surface that prevents fork progression. (B) Cartoon representation of the expected replication intermediates (RIs) at the <i>RuiuR</i> locus as analysed by two-dimensional gel electrophoresis (2DGE). Left panel: RIs expected when the <i>Ase</i>I fragment indicated is replicated passively (no fork arrest at the <i>RTS1</i> barrier). Right panel - RIs expected in <i>RuiuR</i> cells upon fork arrest. (C) Left panel: control cells with no pause, demonstrating the position of the Y-arc. Middle and right panels: The <i>rtf1</i> ORF was inserted at the <i>urg1</i> locus in rtf1Ξ cells by Cre-mediated cassette exchange to create YSM098 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083800#pone-0083800-t001" target="_blank">Table 1</a>). The strain was grown in EMM+LA at 30Β°C (asynchronous culture) and Rtf1 protein induced by the addition of uracil at 0.25 mg/ml. Samples taken at time-points shown. Chromosomal DNA was digested by A<i>se</i>I, and RIs were analysed by 2DGE.</p
Principals of RCME and plasmids created.
<p>(<b>A</b>). Schematic showing the process of RCME (Watson 2008): (i) starting with a base strain in which the <i>urg1</i> ORF is replaced by an antibiotic marker (each of <i>hphMX6</i>, <i>natMX6</i> and <i>kanMX6</i> are available) that is flanked by (incompatible) loxP and loxM3 sites, a plasmid (ii) is introduced. This plasmid contains the cloned gene of interest and any tagging sequences positioned between loxP and loxM3 sites. It also expresses Cre recombinase. Site-directed recombination next exchanges the sequences between the plasmid and the chromosome (iii). Successful exchange can easily be identified by loss of the antibiotic marker, typically seen in greater than 50% of cells. Plasmid loss in these colonies is then confirmed by replica plating to verify colonies are leu<sup>β</sup>. In our experience, all of these are successful integrants. (<b>B</b>). Plasmid for expression of untagged sequences (NO DSR) as previously published <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083800#pone.0083800-Watson1" target="_blank">[7]</a>. Shown is a schematic of the sequence between loxP (P) and loxM3 (M3) for pAW8E<i>Nde</i>I. A start codon is formed from an <i>Nde</i>I site. (<b>C</b>) Equivalent schematic of pAW8E<i>Nde</i>I containing various DSR sequences. (<b>D</b>) Schematic of plasmid used to express proteins with either a yEGFP tag, a 3xHA tag or an HA combined with an IAA17 degron tag (HAIAA17) (all with NO DSR). L β=β poly-tyrosineβglycineβserine (TGS) linker: TAG β=β yEGFP, 3xHA or HAIAA17 protein tag. (<b>E</b>) Equivalent schematic of pAW8E<i>Nde</i>I C-terminal tagging plasmids that also contain various DSR sequences. HA β=β human influenza hemagglutinin protein tag, yEGFP β=β yeast codon optimised green fluorescent protein, HAIAA17 β=β Degron from Arabidopsis thaliana transcription repressor.</p
DSR activity reduces both induced and repressed P<i><sub>urg1lox</sub></i> protein levels.
<p>(<b>A</b>) Schematic illustration outlining the repair of a single HO-induced DSB in a G2 phase <i>S. pombe</i> cell. Repair in a normal (WT) cell can occur by either by homologous recombination (HR) or single strand annealing (SSA) (left panel) whereas repair in an HR deficient <i>rhp51</i>-delete cell can only occur via SSA (right panel). Grey box β=β region of homology, HOcs β=β HO endonuclease cut site. (<b>B</b>). Steady-state rate of <i>his3</i><sup>+</sup> marker loss in WT HR proficient cells compared to HR deficient <i>rhp51</i>-delete cells. The HO endonuclease ORF tagged at the C-terminus with yEGFP was inserted by Cre-mediated cassette exchange into the <i>urg1</i> locus in WT cells containing the HOcs-HIS construct to create AW741 (NO DSR), AW743 (3XDSR), AW745 (4XDSR), AW747 (6XDSR), AW749 (8XDSR) and AW751 (<i>spo5</i>DSR) and in <i>rhp51</i>-delete cells to create AW734 (NO DSR), AW816 (3XDSR), AW818 (4XDSR), AW820 (6XDSR), AW822 (8XDSR) and AW739 (<i>spo5</i>DSR). Logarithmically growing cells cultured in EMM+L were plated onto EMM+LH plates and grown at 30Β°C. Colonies were replica plated onto EMM+L plates and the percentage of histidine auxotrophic (his<sup>β</sup>) cells calculated. The assay was repeated at least three times and the average numbers are presented as the mean +/β SD. (<b>C</b>) The kinetics of HO-cyEGFP protein accumulation is unaffected by DSR activity. Time-course showing accumulation of HO-cyEGFP protein levels following induction of P<i><sub>urg1lox</sub></i>. Logarithmically growing AW671 (NO DSR) and AW673 (<i>spo5</i>DSR) cells (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083800#pone-0083800-t001" target="_blank">Table 1</a>) were induced by the addition of uracil at 0.25mg/ml. HO-cyEGFP protein levels were examined by western blot analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083800#pone-0083800-g002" target="_blank">Figure 2B</a>. Samples taken at time points shown (mins). (<b>D</b>) DSR activity slows <i>his3+</i> marker loss in the P<i><sub>urg1lox</sub></i>-HO/HOcs-HIS SSA assay. Strains AW741 (NO DSR), AW743 (3XDSR), AW745 (4XDSR), AW747 (6XDSR), AW749 (8XDSR) and AW751 (<i>spo5</i>DSR) were grown in EMM+L to mid-log phase and uracil added at 0.25 mg/ml to induce P<i><sub>urg1lox</sub></i>. Cells were plated onto EMM+LH plates and grown at 30Β°C. Colonies were replica plated onto EMM+L plates and the percentage of histidine auxotrophic cells calculated. Samples taken at time-points shown (mins). The assay was repeated twice and numbers shown are the mean. X axis β=β percentage of histidine auxotrophic cells, Y-axis β=β time in minutes.</p