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Yeast recombination pathways triggered by topoisomerase II-mediated DNA breaks
Michelle Sabourin1, John L Nitiss, Karin C Nitiss
1Department of Biochemistry,Vanderbilt University School of Medicine, Nashville, TN 37232-0146, USA.
Abstract:
Topoisomerase II is a ubiquitous enzyme that removes knots and tangles from the genetic material by generating transient double-strand DNA breaks. While the enzyme cannot perform its essential cellular functions without cleaving DNA, this scission activity is inherently dangerous to chromosomal integrity. In fact, etoposide and other clinically important anticancer drugs kill cells by increasing levels of topoisomerase II-mediated DNA breaks. Cells rely heavily on recombination to repair double-strand DNA breaks, but the specific pathways used to repair topoisomerase II-generated DNA damage have not been defined. Therefore, Saccharomyces cerevisiae was used as a model system to delineate the recombination pathways that repair DNA breaks generated by topoisomerase II. Yeast cells that expressed wild-type or a drug-hypersensitive mutant topoisomerase II or overexpressed the wild-type enzyme were examined. Based on cytotoxicity and recombination induced by etoposide in different repair-deficient genetic backgrounds, double-strand DNA breaks generated by topoisomerase II appear to be repaired primarily by the single-strand invasion pathway of homologous recombination. Non-homologous end joining also was triggered by etoposide treatment, but this pathway was considerably less active than single-strand invasion and did not contribute significantly to cell survival in S.cerevisiae.
Insights
Topoisomerase II (a DNA-repair enzyme) creates DNA breaks essential for its function, but anticancer drugs like etoposide exploit this. Yeast studies show homologous recombination, specifically single-strand invasion, is the main repair pathway for these breaks.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Topoisomerase II enzymes are crucial for managing DNA topology, performing essential functions by creating transient DNA double-strand breaks.
- While vital, these breaks can compromise chromosomal integrity, especially when exacerbated by anticancer agents like etoposide.
- Understanding the repair mechanisms for topoisomerase II-induced DNA damage is critical for cancer therapy and DNA repair research.
Purpose of the Study:
- To identify the specific DNA repair pathways responsible for resolving topoisomerase II-generated double-strand breaks.
- To investigate the role of homologous recombination and non-homologous end joining in repairing etoposide-induced DNA damage in Saccharomyces cerevisiae.
Main Methods:
- Utilized Saccharomyces cerevisiae (yeast) as a model organism.
- Examined yeast strains expressing wild-type, drug-hypersensitive mutant, or overexpressed topoisomerase II.
- Assessed cytotoxicity and recombination frequencies following etoposide treatment in various DNA repair-deficient mutant backgrounds.
Main Results:
- Etoposide treatment induced significant DNA double-strand breaks mediated by topoisomerase II.
- The single-strand invasion pathway of homologous recombination was identified as the predominant repair mechanism for these breaks.
- Non-homologous end joining was also activated but contributed minimally to overall cell survival.
Conclusions:
- The study elucidates that homologous recombination, particularly single-strand invasion, is the primary pathway for repairing topoisomerase II-mediated DNA double-strand breaks in yeast.
- Non-homologous end joining plays a minor role in the repair of such damage and offers limited contribution to cell survival.
- These findings provide crucial insights into DNA repair strategies and the mechanisms targeted by topoisomerase II-based anticancer drugs.