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Human topoisomerase I mediates illegitimate recombination leading to DNA insertion into the ribosomal DNA locus in Saccharomyces cerevisiae

Abstract

Eukaryotic type I DNA topoisomerases catalyze the relaxation of supercoiled DNA, and play a critical role in DNA replication, transcription and recombination. They are highly conserved, both in sequence and mechanism of activity, from yeast to mammalian cells. We tested the effect of human topoisomerase I (hTOP1) on illegitimate insertion in yeast by expressing the hTOP1 gene in top1Δ yeast (ytop1Δ) cells. hTOP1 increased the frequency of illegitimate recombination into genomic DNA by 20- to 90-fold relative to the level in ytop1Δ cells, while it had no effect on homologous integration. The addition of the topoisomerase I inhibitor camptothecin blocked this increase in the level of illegitimate insertion. The expression of hTOP1 also significantly enhanced the fraction of integration events in ribosomal DNA (rDNA)—from 16% to 60%, indicating that the rDNA is a highly preferred target for hTOP1. Integrations occurred at the consensus sequence 5′ (T/A) (G/C/A) (T/A) (T/C/A) 3′ in hTOP1 expressing cells. A similar preferred break-site consensus sequence was previously identified in vitro for topoisomerases from rat liver and wheat germ.

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Acknowledgements

We thank Mary-Ann Bjornsti and Dan Gietz for plasmids and strains. We also thank members of the Schiestl lab for comments on the manuscript. This research was supported by Grant No. CN-83 from the American Cancer Society and Research Career Development Award No. ES00299 from the National Institute of Environmental Health Sciences, as well as funding from the UCLA Center for Occupational and Environmental Health to RHS.

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Correspondence to R. H. Schiestl.

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Communicated by R. Devoret

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Zhu, J., Schiestl, R.H. Human topoisomerase I mediates illegitimate recombination leading to DNA insertion into the ribosomal DNA locus in Saccharomyces cerevisiae . Mol Genet Genomics 271, 347–358 (2004). https://doi.org/10.1007/s00438-004-0987-7

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Keywords

  • Human topoisomerase I
  • Genomic target sites
  • Genomic distribution
  • Nonhomologous recombination
  • Ribosomal DNA