Checkpoint effects and telomere amplification during DNA re-replication in fission yeast

<p>Abstract</p> <p>Background</p> <p>Although much is known about molecular mechanisms that prevent re-initiation of DNA replication on newly replicated DNA during a single cell cycle, knowledge is sparse regarding the regions that are most susceptible to re-replication when those mechanisms are bypassed and regarding the extents to which checkpoint pathways modulate re-replication. We used microarrays to learn more about these issues in wild-type and checkpoint-mutant cells of the fission yeast, <it>Schizosaccharomyces pombe</it>.</p> <p>Results</p> <p>We found that over-expressing a non-phosphorylatable form of the replication-initiation protein, Cdc18 (known as Cdc6 in other eukaryotes), drove re-replication of DNA sequences genome-wide, rather than forcing high level amplification of just a few sequences. Moderate variations in extents of re-replication generated regions spanning hundreds of kilobases that were amplified (or not) ~2-fold more (or less) than average. However, these regions showed little correlation with replication origins used during S phase. The extents and locations of amplified regions in cells deleted for the checkpoint genes encoding Rad3 (ortholog of human ATR and budding yeast Mec1) and Cds1 (ortholog of human Chk2 and budding yeast Rad53) were similar to those in wild-type cells. Relatively minor but distinct effects, including increased re-replication of heterochromatic regions, were found specifically in cells lacking Rad3. These might be due to Cds1-independent roles for Rad3 in regulating re-replication and/or due to the fact that cells lacking Rad3 continued to divide during re-replication, unlike wild-type cells or cells lacking Cds1. In both wild-type and checkpoint-mutant cells, regions near telomeres were particularly susceptible to re-replication. Highly re-replicated telomere-proximal regions (50–100 kb) were, in each case, followed by some of the least re-replicated DNA in the genome.</p> <p>Conclusion</p> <p>The origins used, and the extent of replication fork progression, during re-replication are largely independent of the replication and DNA-damage checkpoint pathways mediated by Cds1 and Rad3. The fission yeast pattern of telomere-proximal amplification adjacent to a region of under-replication has also been seen in the distantly-related budding yeast, which suggests that subtelomeric sequences may be a promising place to look for DNA re-replication in other organisms.</p

Checkpoint independence of most DNA replication origins in fission yeast

<p>Abstract</p> <p>Background</p> <p>In budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, <it>Schizosaccharomyces pombe</it>, we used microarrays to investigate the use of origins by wild-type and checkpoint-mutant strains in the presence of hydroxyurea (HU), which limits the pool of deoxyribonucleoside triphosphates (dNTPs) and activates the replication checkpoint. The checkpoint-mutant cells carried deletions either of <it>rad3 </it>(which encodes the fission yeast homologue of ATR) or <it>cds1 </it>(which encodes the fission yeast homologue of Chk2).</p> <p>Results</p> <p>Our microarray results proved to be largely consistent with those independently obtained and recently published by three other laboratories. However, we were able to reconcile differences between the previous studies regarding the extent to which fission yeast replication origins are affected by the replication checkpoint. We found (consistent with the three previous studies after appropriate interpretation) that, in surprising contrast to budding yeast, most fission yeast origins, including both early- and late-firing origins, are not significantly affected by checkpoint mutations during replication in the presence of HU. A few origins (~3%) behaved like those in budding yeast: they replicated earlier in the checkpoint mutants than in wild type. These were located primarily in the heterochromatic subtelomeric regions of chromosomes 1 and 2. Indeed, the subtelomeric regions defined by the strongest checkpoint restraint correspond precisely to previously mapped subtelomeric heterochromatin. This observation implies that subtelomeric heterochromatin in fission yeast differs from heterochromatin at centromeres, in the mating type region, and in ribosomal DNA, since these regions replicated at least as efficiently in wild-type cells as in checkpoint-mutant cells.</p> <p>Conclusion</p> <p>The fact that ~97% of fission yeast replication origins – both early and late – are not significantly affected by replication checkpoint mutations in HU-treated cells suggests that (i) most late-firing origins are restrained from firing in HU-treated cells by at least one checkpoint-independent mechanism, and (ii) checkpoint-dependent slowing of S phase in fission yeast when DNA is damaged may be accomplished primarily by the slowing of replication forks.</p

Checkpoint effects and telomere amplification during DNA re-replication in fission yeast-0

Ine at 0 hours. (A) Fluorescence microscopy of cells 25 hours after induction. Cells are stained with DAPI to show nuclei. Arrows show binucleate Δ cells, which are evidence for active cell division. (B) Histogram plots of DNA content determined by flow cytometry for 0, 17, 21, and 25 hours after removal of thiamine. (C) Density plots of DNA content (fluorescence intensity, x-axis) versus cell size (forward scatter, y-axis) at 25 hours. Wild-type and Δ cells are greatly elongated with high DNA content whereas Δ cells are shorter and have lower DNA content due to cell division during re-replication. The insets show the number of cells out of 10,000 represented by pixels of the indicated color.<p><b>Copyright information:</b></p><p>Taken from "Checkpoint effects and telomere amplification during DNA re-replication in fission yeast"</p><p>http://www.biomedcentral.com/1471-2199/8/119</p><p>BMC Molecular Biology 2007;8():119-119.</p><p>Published online 21 Dec 2007</p><p>PMCID:PMC2265721.</p><p></p

Checkpoint effects and telomere amplification during DNA re-replication in fission yeast-3

Hromosomes 1 and 2 in fission yeast or for all chromosomes in budding yeast (E). Chromosome 3, which has atypical telomeres due to the presence of rDNA repeats, was omitted from the analysis. Probe values for wild-type (A; green), Δ (B; blue), Δ (C; red), and all three strains (D; green, blue, and red) induced for Cdc18* over-expression at 17, 21, and 25 hours of Cdc18* induction are shown. Re-replication is enhanced up to 50 kb from the ends of telomeres in wild-type and Δ cells and up to 100 kb in Δ cells. (E)Re-replication of sub-telomeric regions up to 50 kb from the ends of chromosomes was also enhanced in checkpoint-competent cells as shown in this figure from Tanny . The figure shows relative enrichment for each spot on their microarray plotted as a function of its distance to the closest telomere for both the re-replicating strain (black) and wild-type strain (gray) [14].<p><b>Copyright information:</b></p><p>Taken from "Checkpoint effects and telomere amplification during DNA re-replication in fission yeast"</p><p>http://www.biomedcentral.com/1471-2199/8/119</p><p>BMC Molecular Biology 2007;8():119-119.</p><p>Published online 21 Dec 2007</p><p>PMCID:PMC2265721.</p><p></p

Checkpoint effects and telomere amplification during DNA re-replication in fission yeast-5

Ine at 0 hours. (A) Fluorescence microscopy of cells 25 hours after induction. Cells are stained with DAPI to show nuclei. Arrows show binucleate Δ cells, which are evidence for active cell division. (B) Histogram plots of DNA content determined by flow cytometry for 0, 17, 21, and 25 hours after removal of thiamine. (C) Density plots of DNA content (fluorescence intensity, x-axis) versus cell size (forward scatter, y-axis) at 25 hours. Wild-type and Δ cells are greatly elongated with high DNA content whereas Δ cells are shorter and have lower DNA content due to cell division during re-replication. The insets show the number of cells out of 10,000 represented by pixels of the indicated color.<p><b>Copyright information:</b></p><p>Taken from "Checkpoint effects and telomere amplification during DNA re-replication in fission yeast"</p><p>http://www.biomedcentral.com/1471-2199/8/119</p><p>BMC Molecular Biology 2007;8():119-119.</p><p>Published online 21 Dec 2007</p><p>PMCID:PMC2265721.</p><p></p

Checkpoint effects and telomere amplification during DNA re-replication in fission yeast-4

S at 25 hours after thiamine removal of wild-type cells (green) and Δ cells (red); the replication profile of wild-type cells replicating under normal conditions in the absence of HU; and the replication profile of wild-type cells replicating in the presence of HU. No smoothing of data was performed. The overall efficiencies of origin firing during HU treatment, from the studies by Mickle . [31], are plotted as black sticks below the HU-arrested replication profile. The lengths of the sticks represent the levels of efficiency of the origins during the HU treatment. The longer the stick, the more efficiently the origin fired. Note that the measure of overall efficiency employed by Mickle . combined efficiency in wild-type cells with efficiencies in checkpoint-mutant cells [31]. For this reason, telomeric origins in chromosomes 1 and 2 show relatively high efficiencies, even though the extents of replication at telomeres were small in wild-type cells. The bottom panel shows the cumulative sum of origin scores in a sliding 100-kb window. Centromeres are indicated by yellow squares. The region spanning 0.8 kb to 1.8 kb along chromosome 1 is highlighted by a light purple box. (B) A closer look at the highlighted amplified region (light purple box in (A)) shows that the pattern of re-replication is clearly different from patterns of replication. (C) The amount of replication under HU stress was compared to the amount of re-replication by plotting relative copy numbers under HU stress for 4 hours [31] against re-replicated to control DNA ratios for strains 25 hours after thiamine removal. Re-replicated to control DNA ratios and relative copy numbers for all probes were plotted for wild-type, Δ, and Δ. Trendlines and R-squared values are provided in all graphs. The Δ strain displayed a slight correlation, lacking in the wild-type and Δ strains.<p><b>Copyright information:</b></p><p>Taken from "Checkpoint effects and telomere amplification during DNA re-replication in fission yeast"</p><p>http://www.biomedcentral.com/1471-2199/8/119</p><p>BMC Molecular Biology 2007;8():119-119.</p><p>Published online 21 Dec 2007</p><p>PMCID:PMC2265721.</p><p></p

Checkpoint effects and telomere amplification during DNA re-replication in fission yeast-1

A from re-replicating cells against DNA from the same strain prior to re-replication. DNA re-replication profiles of wild-type cells induced for 17 hours, 21 hours, and 25 hours are shown for chromosomes 1 (A), 2 (B), and 3 (C). Centromeres are indicated by yellow squares. No smoothing of data has been applied. Vertical lines indicate the relative level of DNA amplification across the genome. A value of 1.0 is the average amount of re-replicated DNA for the genome. Values greater than 1.0 represent probes which were replicated more than the average amount of re-replication. The results reveal a gradual increase in differences between the most amplified and least amplified regions over time.<p><b>Copyright information:</b></p><p>Taken from "Checkpoint effects and telomere amplification during DNA re-replication in fission yeast"</p><p>http://www.biomedcentral.com/1471-2199/8/119</p><p>BMC Molecular Biology 2007;8():119-119.</p><p>Published online 21 Dec 2007</p><p>PMCID:PMC2265721.</p><p></p

Checkpoint independence of most DNA replication origins in fission yeast-5

<p><b>Copyright information:</b></p><p>Taken from "Checkpoint independence of most DNA replication origins in fission yeast"</p><p>http://www.biomedcentral.com/1471-2199/8/112</p><p>BMC Molecular Biology 2007;8():112-112.</p><p>Published online 19 Dec 2007</p><p>PMCID:PMC2235891.</p><p></p>. The position of the centromere on each chromosome is indicated by a light yellow rectangle. The positions of origins classified as strong, medium, weak or very weak are identified by vertical lines. The lines range in color from red (strong) to brown (very weak) and from long (strong) to short (very weak). The positions of potential origins below the detection limit are indicated by the text character, "0", and ambiguous origins (where our probes were too widely spaced to permit confident evaluation) are shown by the character, "?". Small circles above each chromosome line indicate the positions of origins identified by Heichinger ([14]; top row of circles; light blue), AT islands (next row of circles; magenta), origins identified by Feng [34] in Δ cells (next row; orange) or in wild-type cells (next row; purple), and pre-RCs identified by Hayashi ([15]; bottom row; dark blue or red circles or squares). For the pre-RCs, the colors blue and red distinguish the pre-RCs that are late/weak or early/strong, respectively. The circles represent pre-RCs that are not affected by deletion of , while the squares indicate pre-RCs that replicate to a greater extent in Δ cells than in wild-type cells [15]. The positions of origins where the signals (our measurements; Additional Files , , ) for both checkpoint-mutant strains were significantly greater than the signal for wild-type cells are indicated by the text character, "C", and the positions of origins with the opposite characteristic (wild-type signal significantly greater than the signals from both checkpoint-mutant strains) are shown by the text character, "W". A pale green background indicates a large region with a high frequency of stronger origins. A pale yellow background indicates a large region with a high frequency of weaker origins. (A) chromosome 1; (B) chromosome 2; (C) chromosome 3