Genome-Destabilizing Effects Associated with Top1 Loss or Accumulation of Top1 Cleavage Complexes in Yeast

<div><p>Topoisomerase 1 (Top1), a Type IB topoisomerase, functions to relieve transcription- and replication-associated torsional stress in DNA. We investigated the effects of Top1 on genome stability in <i>Saccharomyces cerevisiae</i> using two different assays. First, a sectoring assay that detects loss of heterozygosity (LOH) on a specific chromosome was used to measure reciprocal crossover (RCO) rates. Features of individual RCO events were then molecularly characterized using chromosome-specific microarrays. In the second assay, cells were sub-cultured for 250 generations and LOH was examined genome-wide using microarrays. Though loss of Top1 did not destabilize single-copy genomic regions, RCO events were more complex than in a wild-type strain. In contrast to the stability of single-copy regions, sub-culturing experiments revealed that <i>top1</i> mutants had greatly elevated levels of instability within the tandemly-repeated ribosomal RNA genes (in agreement with previous results). An intermediate in the enzymatic reaction catalyzed by Top1 is the covalent attachment of Top1 to the cleaved DNA. The resulting Top1 cleavage complex (Top1cc) is usually transient but can be stabilized by the drug camptothecin (CPT) or by the <i>top1-T722A</i> allele. We found that increased levels of the Top1cc resulted in a five- to ten-fold increase in RCOs and greatly increased instability within the rDNA and <i>CUP1</i> tandem arrays. A detailed analysis of the events in strains with elevated levels of Top1cc suggests that recombinogenic DNA lesions are introduced during or after DNA synthesis. These results have important implications for understanding the effects of CPT as a chemotherapeutic agent.</p></div

CPT treatment and Top1-T722A expression increase RCOs during or after DNA synthesis.

<p>Rates of red/white sectoring associated with <b>(A)</b> CPT treatment or <b>(B)</b> Top1-T722A expression are shown. The cell cycle phase when an RCO-initiating event occurred was inferred by the associated gene conversion pattern: <b>(C)</b> CPT treatment or <b>(D)</b> Top1-T722A expression stimulates initiation primarily in G2. Black, G1-associated; gray, G2-associated; white, simple CO (no GC).</p

Distributions of LOH breakpoints.

<p>Plotted is the number of times each SNP was included in a crossover-associated conversion tract (Y-axis) and the SGD coordinates for chromosome IV (X-axis) in <b>(A)</b> WT, <b>(B)</b><i>top1Δ</i>/<i>top1Δ</i>, <b>(C)</b> WT + CPT, and <b>(D)</b><i>top1-T722A</i> sectors. Hotspots 4 (HS4: Chr IV SGD coordinates 970-1000kb) and 7 (HS7: Chr IV SGD coordinates 1260-1270kb) were previously noted in a WT background [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005098#pgen.1005098.ref018" target="_blank">18</a>] are indicated by the shaded region.</p

CPT treatment and Top1-T722A expression result in increased RCOs at the rDNA locus.

<p>An rDNA-distal PCR product from sub-culture 10 clones was digested with <i>Spe</i>I and analyzed by gel electrophoresis. Dotted, heterozygous; black, homozygous for W303-1A; white, homozygous for YJM789.</p

<i>CUP1</i> instability associated with CPT treatment or Top1-T722A expression.

<p>Panel <b>(A)</b> illustrates an increase in the number of <i>CUP1</i> repeats on the W303-1A allele (red peak in boxed region) as detected by microarrays. In <b>(B)</b> a representative <i>CUP1</i> Southern blot is shown. DNA isolated from cells sub-cultured 10 times (~250 generations) was digested with <i>Eco</i>RI and the size of the array on each homolog was examined by Southern blot using a <i>CUP1</i>-specific probe. The arrows indicate the sizes of the <i>CUP1</i> arrays in the parent. The most robust band was used as the measure for gain or loss. <b>(C)</b> CPT treatment or Top1-T722A expression lead to a significant increase in copy number variation (CNV) after 10 sub-cultures. Also shown is the percentage of alleles that lose (black bars) compared to alleles that gained <i>CUP1</i> repeats (white bars).</p

Mechanisms of CNV at the <i>CUP1</i> locus.

<p><b>(A)</b> An unequal crossover between two misaligned sister chromatids results in one chromatid gaining repeats and the other losing repeats. This event can occur between sister chromatids or homologous chromosomes. <b>(B)</b> Break-induced replication (BIR). A DSB can be repaired using the sister chromatid or homolog as a template. Replication to the end of the template chromosome restores the broken chromosome and the template remains unchanged. <b>(C)</b> Gene conversion (GC). Repair of a DSB with either the sister chromatid or homologous chromosome by GC results in the repaired chromatid gaining (or losing) copies of the <i>CUP1</i> repeat while the donor chromatid remains the same. <b>(D)</b> Single strand annealing (SSA). A double strand break (DSB) can be resected to a region of homology and annealing/rejoining of ends leads to a loss of <i>CUP1</i> repeats. <b>(E)</b> An intrachromatid exchange that involves repeats on the same chromatid results in a loss of multiple <i>CUP1</i> repeats.</p

Detecting and characterizing RCOs on chromosome IV.

<p><b>(A)</b> JSC25 chromatids shown in red are derived from W303-1A and contain a <i>KANMX</i> cassette on the right arm of chromosome IV. Chromatids in black are derived from YJM789 and contain a <i>SUP4-o</i> marker on the right arm of chromosome IV. In addition the diploid strain is homozygous for the <i>ade2-1</i> mutation. An RCO that leads to LOH generates a red/white sectored colony (white arrow) when the daughter cells segregate. <b>(B)</b> Microarray analysis. The W303-1A-related SNP hybridization signal is shown as a red line and that of YJM789 is the blue line. A hybridization value of ~1 indicates a single copy of a given SNP is present (heterozygous SNP), a value between 1.5 and 2 indicates two copies of the SNP, and a value of 0–0.5 indicates no copy of the SNP is present. Shown is the white side of a sector with a transition from heterozygous to YJM789-homozygous SNPs.</p