Multiple Endonucleases Function to Repair Covalent Topoisomerase I Complexes in Saccharomyces cerevisiae

Department of Radiation Oncology, Stanford University School of Medicine, California 94305-5152, USA.
Genetics (Impact Factor: 5.96). 07/2005; 170(2):591-600. DOI: 10.1534/genetics.104.028795
Source: PubMed


Topoisomerase I plays a vital role in relieving tension on DNA strands generated during replication. However if trapped by camptothecin or other DNA damage, topoisomerase protein complexes may stall replication forks producing DNA double-strand breaks (DSBs). Previous work has demonstrated that two structure-specific nucleases, Rad1 and Mus81, protect cells from camptothecin toxicity. In this study, we used a yeast deletion pool to identify genes that are important for growth in the presence of camptothecin. In addition to genes involved in DSB repair and recombination, we identified four genes with known or implicated nuclease activity, SLX1, SLX4, SAE2, and RAD27, that were also important for protection against camptothecin. Genetic analysis revealed that the flap endonucleases Slx4 and Sae2 represent new pathways parallel to Tdp1, Rad1, and Mus81 that protect cells from camptothecin toxicity. We show further that the function of Sae2 is likely due to its interaction with the endonuclease Mre11 and that the latter acts on an independent branch to repair camptothecin-induced damage. These results suggest that Mre11 (with Sae2) and Slx4 represent two new structure-specific endonucleases that protect cells from trapped topoisomerase by removing topoisomerase-DNA adducts.

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Available from: Changchun Deng, May 13, 2015
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    • "Interestingly, the two outlier cell lines (H69 and H187) that display relatively high sensitivity to topotecan possess remarkably higher levels of TDP1 than the rest. It is worth noting that TDP1 is not the only factor involved in the cellular response to topotecan and that redundant and partially overlapping pathways driven by TDP2, MUS81, XPF-ERCC1, and MRE11 are also implicated in this process [27,38,39]. It is possible that the H69 and H187 cell lines lack one or more TDP1 alternative pathway for dealing with TOP1-breaks, which might account for their unexpected high sensitivity to topotecan. "
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    ABSTRACT: Background and objective Small cell lung cancer (SCLC) is one of the most challenging tumors to treat due to high proliferation rate, early metastatic dissemination and rapid development of chemotherapy resistance. The current treatment protocols involve the use of topoisomerase 1 (TOP1) poisons such as irinotecan and topotecan in combination with platinum-based compounds. TOP1 poisons kill cancer cells by trapping TOP1 on DNA, generating lethal DNA double-strand breaks. A potential mechanism employed by cancer cells to resist killing by TOP1 poisons is to overexpress enzymes involved in the repair of TOP1-DNA breaks. Tyrosyl DNA phosphodiesterase 1 (TDP1) is a key player in this process and despite its importance, no data is currently available to correlate TDP1 protein and mRNA levels with catalytic activity in SCLC. In addition, it is not known if TDP1 and TOP1 protein levels correlate with the cellular response of SCLC to TOP1 based therapies. Methods and results We report a remarkable variation in TDP1 and TOP1 protein levels in a panel of SCLC cell lines. TDP1 protein level correlates well with TDP1 mRNA and TDP1 catalytic activity, as measured by two newly developed independent activity assays, suggesting the potential utility of immunohistochemistry in assessing TDP1 levels in SCLC tissues. We further demonstrate that whilst TDP1 protein level alone does not correlate with topotecan sensitivity, TDP1/TOP1 ratio correlates well with sensitivity in 8 out of 10 cell lines examined. Conclusion This study provides the first cellular analyses of TDP1 and TOP1 in SCLC and suggests the potential utility of TDP1/TOP1 ratio to assess the response of SCLC to topotecan. The establishment and validation of an easy-to-use TDP1 enzymatic assay in cell extracts could be exploited as a diagnostic tool in the clinic. These findings may help in stratifying patients that are likely to benefit from TOP1 poisons and TDP1 inhibitors currently under development.
    Full-text · Article · Jul 2014 · Journal of Cancer Science and Therapy
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    • "Slx4 appears to act as a scaffold that interacts with several other nucleases and allows for the assembly of complexes that may be specific for aberrant DNA structures (Munoz et al. 2009). Slx1/Slx4 deficient mutant are camptothecin hypersensitive (Deng et al. 2005), and are also hypersensitive to Top2-targeting drugs (see Table 18.1 and the discussion in Sect. 18.7). "
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    ABSTRACT: 18.1 Introduction Topoisomerase II (Top2) is an important anticancer drug target. Agents such as etoposide and doxorubicin are broadly used in a wide variety of malignancies (Baldwin and Osheroff 2005; Choi et al. 2008; Dombernowsky et al. 1996; Lieu et al. 2009; Verborg et al. 2008; Walker and Nitiss 2002). Most drugs that target Top2 generate DNA damage as a direct consequence of the catalytic activity of the enzyme. All topoisomerases cleave DNA by forming a covalent complex between the enzyme and DNA, and agents that perturb the catalytic cycle have the potential to trap the enzyme and introduce DNA damage. The DNA damage caused by interfering with a topoisomerase is unique because it includes both DNA strand breaks and protein covalently bound to DNA. Agents that lead to the trapping of topoisomerases on DNA have been termed topoisomerase poisons to highlight the importance of cellular damage induced by these agents. Because topoisomerase poisons lead to cell killing largely through enzyme-mediated damage, pathways that repair this damage are critical determinants of clinical response to these agents. A major goal of this chapter is to highlight our current understanding of how DNA repair pathways affect sensitivity to Top2 poisons. It is hoped that some of these concepts will lead to new approaches for the clinical application of Top2 targeting agents. In addition to the clinical importance of Top2 targeting agents, these drugs have served as model compounds to study cellular responses to DNA damage. Unlike ionizing radiation or alkylating agents, many Top2 targeting drugs are highly specific,
    Full-text · Chapter · Jan 2012
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    • "Because Mus81-Eme1 is a 3 flap endonuclease, it has been postulated that it would cleave 5 from the Top1cc and participate in the excision of trapped Top1 from the DNA (Liu et al., 2002; Vance and Wilson, 2002; Deng et al., 2005). To determine whether Mus81 is involved in Top1cc removal, we analyzed the formation of Top1cc's in WT and Mus81 / cells treated with various CPT concentrations. "
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    ABSTRACT: Deoxyribonucleic acid (DNA) topoisomerases are essential for removing the supercoiling that normally builds up ahead of replication forks. The camptothecin (CPT) Top1 (topoisomerase I) inhibitors exert their anticancer activity by reversibly trapping Top1-DNA cleavage complexes (Top1cc's) and inducing replication-associated DNA double-strand breaks (DSBs). In this paper, we propose a new mechanism by which cells avoid Top1-induced replication-dependent DNA damage. We show that the structure-specific endonuclease Mus81-Eme1 is responsible for generating DSBs in response to Top1 inhibition and for allowing cell survival. We provide evidence that Mus81 cleaves replication forks rather than excises Top1cc's. DNA combing demonstrated that Mus81 also allows efficient replication fork progression after CPT treatment. We propose that Mus81 cleaves stalled replication forks, which allows dissipation of the excessive supercoiling resulting from Top1 inhibition, spontaneous reversal of Top1cc, and replication fork progression.
    Full-text · Article · Nov 2011 · The Journal of Cell Biology
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