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

摘要

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 Saccharomyces cerevisiae 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 top1 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 top1-T722A 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 CUP1 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. Author Summary Topoisomerase I (Top1) nicks one strand of DNA to relieve torsional stress associated with replication, transcription and chromatin remodeling. The enzyme forms a transient, covalent intermediate with the nicked DNA and stabilization of the cleavage complex (Top1cc) leads to genetic instability. We examined the effect of Top1 loss or Top1cc stabilization on genome-wide mitotic stability and on mitotic crossovers that lead to loss of heterozygosity (LOH) in budding yeast. The level of Top1cc was elevated using the chemotherapeutic drug camptothecin or a mutant form of the enzyme. Whereas loss of Top1 only destabilized ribosomal DNA repeats, Top1cc accumulation was additionally associated with elevated LOH and genome-wide instability. In particular, the Top1cc greatly elevated copy number variation at the CUP1 tandem-repeat locus, consistent with elevated sister chromatid recombination. Molecular examination of LOH events associated with the Top1cc was also consistent with generation of recombination-initiating lesions during or after DNA synthesis. These results demonstrate that the use of topoisomerase inhibitors results in widespread genome instability that may contribute to secondary neoplasms.