Department of Immunology, University of Washington School of Medicine, Seattle, Washington, United States of America.
PLoS One. 2010 Oct 28;5(10):e15387. doi: 10.1371/journal.pone.0015387.
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.
喜树碱 (CPT) 和依托泊苷 (ETP) 捕获拓扑异构酶-DNA 共价中间体，导致 DNA 损伤形成，如果未修复则可能具有细胞毒性。CPT 和 ETP 是广泛用于癌症化疗的分子的原型，因此，确定修复这些化合物治疗引起的损伤的机制具有重要意义。在酿酒酵母中，编码高度保守因子的 MRE11 缺乏会极大地增强对 CPT 或 ETP 处理的敏感性。这被认为反映了双链断裂 (DSB) 修复途径在对这些药物的反应中的重要性。在这里，我们报告说，表达 mre11-H59A 等位基因的酿酒酵母菌株，该等位基因在保守的活性位点组氨酸处发生突变，对羟基脲敏感，也对诱导 DSB 的电离辐射敏感，但对 CPT 或 ETP 不敏感。我们表明，编码能够体外释放 5'-和 3'-共价拓扑异构酶-DNA 复合物的酪氨酸-DNA 磷酸二酯酶活性的 TDP1 有助于在 mre11-H59A 背景下对 ETP 产生抗性，但对 CPT 无抗性。我们进一步表明，MRE11 介导的 CPT 和 ETP 抗性独立于 SAE2，因此独立于 MRE11 和 SAE2 在同源定向修复和减数分裂中从 DNA 末端去除 Spo11 的协调功能。这些结果确定了 MRE11 在拓扑异构酶毒物反应中的作用与 DSB 修复或减数分裂 DNA 处理中的作用不同。它们还表明，细胞在修复 DSB 方面的效率可能与对拓扑异构酶毒物的抗性无关，这一发现对于为这些化合物治疗具有特定 DNA 修复缺陷的肿瘤进行分层具有潜在意义。
