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MRE11 function in response to topoisomerase poisons is independent of its function in double-strand break repair in Saccharomyces cerevisiae.

Hamilton NK, Maizels N - PLoS ONE (2010)

Bottom Line: Camptothecin (CPT) and etoposide (ETP) trap topoisomerase-DNA covalent intermediates, resulting in formation of DNA damage that can be cytotoxic if unrepaired.In S. cerevisiae, deficiency in MRE11, which encodes a highly conserved factor, greatly enhances sensitivity to treatment with CPT or ETP.We show that TDP1, which encodes a tyrosyl-DNA phosphodiesterase activity able to release both 5'- and 3'-covalent topoisomerase-DNA complexes in vitro, contributes to ETP-resistance but not CPT-resistance in the mre11-H59A background.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Washington School of Medicine, Seattle, Washington, United States of America.

ABSTRACT
Camptothecin (CPT) and etoposide (ETP) trap topoisomerase-DNA covalent intermediates, resulting in formation of DNA damage that can be cytotoxic if unrepaired. CPT and ETP are prototypes for molecules widely used in chemotherapy of cancer, so defining the mechanisms for repair of damage induced by treatment with these compounds is of great interest. In S. cerevisiae, deficiency in MRE11, which encodes a highly conserved factor, greatly enhances sensitivity to treatment with CPT or ETP. This has been thought to reflect the importance of double-strand break (DSB) repair pathways in the response to these to agents. Here we report that an S. cerevisiae strain expressing the mre11-H59A allele, mutant at a conserved active site histidine, is sensitive to hydroxyurea and also to ionizing radiation, which induces DSBs, but not to CPT or ETP. We show that TDP1, which encodes a tyrosyl-DNA phosphodiesterase activity able to release both 5'- and 3'-covalent topoisomerase-DNA complexes in vitro, contributes to ETP-resistance but not CPT-resistance in the mre11-H59A background. We further show that CPT- and ETP-resistance mediated by MRE11 is independent of SAE2, and thus independent of the coordinated functions of MRE11 and SAE2 in homology-directed repair and removal of Spo11 from DNA ends in meiosis. These results identify a function for MRE11 in the response to topoisomerase poisons that is distinct from its functions in DSB repair or meiotic DNA processing. They also establish that cellular proficiency in repair of DSBs may not correlate with resistance to topoisomerase poisons, a finding with potential implications for stratification of tumors with specific DNA repair deficiencies for treatment with these compounds.

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TDP1 contributes to ETP-resistance of mre11-H59A and mre11-H125N strains.Serial spot dilution assays of sensitivity to ETP of indicated S. cerevisiae derivatives of parental line Jel1; notations as in Fig. 3. Cells were spotted at 10-fold serial dilutions on rich plates containing no drug (left) or indicated concentrations of ETP.
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pone-0015387-g004: TDP1 contributes to ETP-resistance of mre11-H59A and mre11-H125N strains.Serial spot dilution assays of sensitivity to ETP of indicated S. cerevisiae derivatives of parental line Jel1; notations as in Fig. 3. Cells were spotted at 10-fold serial dilutions on rich plates containing no drug (left) or indicated concentrations of ETP.

Mentions: In S. cerevisiae, MRE11-deficiency causes sensitivity to ETP, which traps covalent complexes formed by Topo II with DNA 5′-ends [4]. We therefore tested ETP sensitivity of a panel of MRE11-deficient strains in a spot dilution assay. We found that the Jel1 mre11Δ strain and its derivative expressing mre11-H213Y were extremely sensitive to ETP, while derivatives expressing mre11-H59A or mre11-H125N were as resistant as Jel1 or the Jel1 mre11Δ derivative expressing MRE11 (Fig. 4, above). Thus, expression of mre11-H59A conferred resistant to ETP and CPT, despite marked sensitivity to HU and IR; and expression of mre11-H125N strain conferred resistant to ETP, despite sensitivity to CPT, HU and IR.


MRE11 function in response to topoisomerase poisons is independent of its function in double-strand break repair in Saccharomyces cerevisiae.

Hamilton NK, Maizels N - PLoS ONE (2010)

TDP1 contributes to ETP-resistance of mre11-H59A and mre11-H125N strains.Serial spot dilution assays of sensitivity to ETP of indicated S. cerevisiae derivatives of parental line Jel1; notations as in Fig. 3. Cells were spotted at 10-fold serial dilutions on rich plates containing no drug (left) or indicated concentrations of ETP.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2965672&req=5

pone-0015387-g004: TDP1 contributes to ETP-resistance of mre11-H59A and mre11-H125N strains.Serial spot dilution assays of sensitivity to ETP of indicated S. cerevisiae derivatives of parental line Jel1; notations as in Fig. 3. Cells were spotted at 10-fold serial dilutions on rich plates containing no drug (left) or indicated concentrations of ETP.
Mentions: In S. cerevisiae, MRE11-deficiency causes sensitivity to ETP, which traps covalent complexes formed by Topo II with DNA 5′-ends [4]. We therefore tested ETP sensitivity of a panel of MRE11-deficient strains in a spot dilution assay. We found that the Jel1 mre11Δ strain and its derivative expressing mre11-H213Y were extremely sensitive to ETP, while derivatives expressing mre11-H59A or mre11-H125N were as resistant as Jel1 or the Jel1 mre11Δ derivative expressing MRE11 (Fig. 4, above). Thus, expression of mre11-H59A conferred resistant to ETP and CPT, despite marked sensitivity to HU and IR; and expression of mre11-H125N strain conferred resistant to ETP, despite sensitivity to CPT, HU and IR.

Bottom Line: Camptothecin (CPT) and etoposide (ETP) trap topoisomerase-DNA covalent intermediates, resulting in formation of DNA damage that can be cytotoxic if unrepaired.In S. cerevisiae, deficiency in MRE11, which encodes a highly conserved factor, greatly enhances sensitivity to treatment with CPT or ETP.We show that TDP1, which encodes a tyrosyl-DNA phosphodiesterase activity able to release both 5'- and 3'-covalent topoisomerase-DNA complexes in vitro, contributes to ETP-resistance but not CPT-resistance in the mre11-H59A background.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Washington School of Medicine, Seattle, Washington, United States of America.

ABSTRACT
Camptothecin (CPT) and etoposide (ETP) trap topoisomerase-DNA covalent intermediates, resulting in formation of DNA damage that can be cytotoxic if unrepaired. CPT and ETP are prototypes for molecules widely used in chemotherapy of cancer, so defining the mechanisms for repair of damage induced by treatment with these compounds is of great interest. In S. cerevisiae, deficiency in MRE11, which encodes a highly conserved factor, greatly enhances sensitivity to treatment with CPT or ETP. This has been thought to reflect the importance of double-strand break (DSB) repair pathways in the response to these to agents. Here we report that an S. cerevisiae strain expressing the mre11-H59A allele, mutant at a conserved active site histidine, is sensitive to hydroxyurea and also to ionizing radiation, which induces DSBs, but not to CPT or ETP. We show that TDP1, which encodes a tyrosyl-DNA phosphodiesterase activity able to release both 5'- and 3'-covalent topoisomerase-DNA complexes in vitro, contributes to ETP-resistance but not CPT-resistance in the mre11-H59A background. We further show that CPT- and ETP-resistance mediated by MRE11 is independent of SAE2, and thus independent of the coordinated functions of MRE11 and SAE2 in homology-directed repair and removal of Spo11 from DNA ends in meiosis. These results identify a function for MRE11 in the response to topoisomerase poisons that is distinct from its functions in DSB repair or meiotic DNA processing. They also establish that cellular proficiency in repair of DSBs may not correlate with resistance to topoisomerase poisons, a finding with potential implications for stratification of tumors with specific DNA repair deficiencies for treatment with these compounds.

Show MeSH
Related in: MedlinePlus