DNA Mismatch and Double-Strand Break Repair

长期目标是了解重组过程和DNA错配修复在细胞在药物和辐射诱导的DNA损伤中存活的能力中所起的作用。我们已经获得了不同作用机制的不同试剂的数据，表明DNA双链断裂是暴露于DNA损伤剂的细胞的一个共同分母。这些物质不仅包括自然产生的物质(一氧化氮)、用于治疗肿瘤疾病的物质(如顺铂、链脲佐菌素)，还包括我们环境中的遗传毒物(如甲基化物质)。已知电离辐射会直接导致双链断裂，但其他试剂不会。我们假设药物诱导的双链断裂是通过含有单链缺口的DNA复制形成的，这些单链缺口是由修复过程产生的，导致复制叉子崩溃。重组机制是修复这些双链断裂所必需的，因此对于细胞防御这些药物与其他修复途径和复制重新启动蛋白的结合是极其重要的。为了验证我们的假设，我们建议使用大肠杆菌作为一个模型系统，因为人们对DNA复制、修复和重组的了解比其他任何生物体都多。此外，影响这些过程的突变菌株只在这种有机体中可用。众所周知，DNA错配修复可以使人和大肠杆菌细胞对顺铂和甲基化药物的毒性作用敏感。事实上，已经从患者身上分离出对这些药物具有抗药性的肿瘤细胞，并显示出错配修复能力不足。我们假设错配修复在耐药中的作用是因为它不再干扰药物诱导的损伤的重组修复。第一个特定目标将测试假设，即在暴露于一氧化氮、甲基化试剂和顺铂后，染色体DNA会形成双链断裂，并以伽马辐射为对照。我们将使用物理方法来检测此类中断。在第二个目标中，将使用一种简单的重组试验来监测上述制剂和伽马辐射诱导的双链断裂水平。我们还将确定药物诱导重组所需的基因产物。第三个目标是用纯化的蛋白质和底物进行生化分析，以检验错配修复干扰重组的假设。如果这些实验支持我们的模型，它将开辟一种通过靶向重组蛋白来对抗肿瘤细胞的新方法。

The long-term objective is to understand the role of recombination processes and DNA mismatch repair in the ability of cells to survive drug and radiation-induced DNA damage. We have obtained data with various agents with different mechanisms of action suggesting that DNA double-strand breaks are a common denominator in cells exposed to DNA damaging agents. These agents include not only those occurring naturally (nitric oxide), those used in the therapy of neoplastic disease (e.g., cisplatin, streptozotocin) but also genotoxicants in our environment (e.g.,methylating agents). Ionizing radiation is known to produce double-strand breaks directly but the other agents do not. We hypothesize that drug-induced double-strand breaks are formed by replication through DNA containing single-strand nicks produced by repair processes leading to collapse of the replication fork. Recombination mechanisms are required to repair these double-strand breaks and are therefore extremely important for cellular defenses against these agents in combination with other repair pathways and replication restart proteins. To test our hypothesis we propose to use E. coli as a model system as more is known about DNA replication, repair and recombination than any other organism. Furthermore, mutant strains affecting these processes are available only in this organism. DNA mismatch repair is known to sensitize human and E. coli cells to the toxic effects of cisplatin and methylating agents. Indeed tumor cells resistant to these agents have been isolated from patients and shown to be deficient in mismatch repair. We hypothesize that the role of mismatch repair in drug- resistance is because it no longer interferes with recombinational repair of drug-induced damage. The first specific aim will test the hypothesis that double-strand breaks are formed in chromosomal DNA after exposure to nitric oxide, methylating agents and cisplatin using gamma-radiation as the control. We will use physical methods to detect such breaks. In the second aim, a simple recombination assay will be used to monitor the level of double-strand breaks induced by the agents listed above and by gamma radiation. We will also determine the gene products required for drug-induced recombination. The third aim will use a biochemical assay with purified proteins and substrates to test the hypothesis that mismatch repair interferes with recombination. If these experiments support our model, it will open up a new approach to combat tumor cells by targeting recombination proteins.