Abstract
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.
Highlights
DNA is a dynamic molecule with ever-changing states of supercoiling, catenation and single/ double strandedness
We examined the effect of Topoisomerase 1 (Top1) loss or Top1 cleavage complex (Top1cc)
Genome-Destabilization Caused by Top1cc Accumulation in Yeast stabilization on genome-wide mitotic stability and on mitotic crossovers that lead to loss of heterozygosity (LOH) in budding yeast
Summary
DNA is a dynamic molecule with ever-changing states of supercoiling, catenation and single/ double strandedness. Underwound (negatively supercoiled) DNA is produced behind RNA polymerases, generating single-stranded DNA that is vulnerable to breakage and promotes formation of RNA:DNA hybrids (R-loops) that inhibit transcription elongation [2]. Chromosomes become catenated via structures such as double Holliday junctions during homologous recombination, and sister chromatids are catenated in the wake of replication. Topoisomerases regulate these topological challenges by breaking the phosphodiester backbone via nucleophilic attack, relieving the topological stress by rotation or strand passage, and re-ligating the DNA by a reverse reaction [3]. In the rDNA of top1Δ mutants, mitotic recombination is increased, transcription is disrupted, the rDNA-containing chromosome migrates aberrantly in gels, and extrachromosomal rDNAcontaining circles are produced [5, 8, 9]
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.