DNA Repair of Multiply Damaged Sites in Cells

细胞内多重损伤位点的 DNA 修复

• 批准号: 7142012
• 负责人: LYNN HARRISON
• 金额: $ 21.94万
• 依托单位: LOUISIANA STATE UNIV HSC SHREVEPORT
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-13 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7142012
• 关键词: DNA damage; DNA directed RNA polymerase; DNA repair; electroporation; enzyme activity; genetic transcription; ionizing radiation; luciferin monooxygenase; oligonucleotides; plasmids; polymerase chain reaction; radiation genetics; reporter genes; southern blotting; tissue /cell culture; transfection /expression vector

DESCRIPTION (provided by applicant): Multiply damaged sites (MDSs) consisting of DMA base damage and strand breaks are produced in cells by certain chemotherapies and radiotherapy. MDSs generated by ionizing radiation can consist of 2-6 damages within < 20 bp and are believed to be more lethal than single lesions. We hypothesize that DNA repair enzymes can convert MDSs to double strand breaks (DSBs), but that non-homologous end- joining (NHEJ) in human cells acts to prevent this conversion of repair intermediates into lethal events. We have developed assays where synthetic MDSs are placed within the luciferase coding region of a plasmid. The plasmid is transferred to cells and a loss of reporter activity, destruction of the plasmid or the introduction of deletions indicates DSB formation. We will utilize these assays in bacteria, mouse embryonic fibroblasts (MEFs) and human fibroblasts proficient and deficient in base excision repair (BER) or non-homologous end-joining (NHEJ). Our first goal (Specific Aims 1 and 2) is to identify bacterial and mammalian DNA repair enzymes that can convert MDSs to DSBs. We will use defined MDSs that simulate ionizing radiation damage to assess the ability of the bacterial AP endonucleases, Fpg and Ogg1/Neil1 to convert MDSs to DSBs. MDSs will be examined that contain 2 or 3 lesions. We hypothesize that the bacterial AP endonucleases will be able to cleave closely opposed AP sites, even in situations where there is near-by base damage, making them prime candidates to sensitize tumor cells to cancer treatments. We have determined that unlike E.coli, mammalian cells do not readily convert MDSs to DSBs and hypothesize that NHEJ is the cause of the difference in the biological outcome of MDSs in the two cell types. By using mammalian cells deficient in the NHEJ proteins, and E.coli that are manipulated to express the prokaryote NHEJ system, our second goal (aim 3) is to test whether the NHEJ proteins prevent the conversion of MDSs to DSBs. We will also identify which NHEJ protein is involved in this DSB avoidance mechanism. In summary, this work will provide basic knowledge that is required to design better cancer treatments. We will identify candidate DNA repair enzymes that can be over-expressed in tumor cells using a gene therapy approach to enhance the lethality of ionizing radiation and chemotherapy, and determine whether it is necessary to disable NHEJ to improve the lethality of the gene therapy.

描述（由申请人提供）：由DMA碱基损伤和链断裂组成的多重损伤位点（MDS）是通过某些化疗和放疗在细胞中产生的。电离辐射产生的 MDS 可能由 < 20 bp 内的 2-6 个损伤组成，并且被认为比单个损伤更致命。我们假设 DNA 修复酶可以将 MDS 转化为双链断裂 (DSB)，但人类细胞中的非同源末端连接 (NHEJ) 会阻止修复中间体转化为致命事件。我们开发了将合成 MDS 置于质粒荧光素酶编码区内的测定方法。将质粒转移到细胞中，报告基因活性丧失、质粒破坏或引入缺失表明 DSB 形成。我们将在碱基切除修复（BER）或非同源末端连接（NHEJ）方面熟练和缺乏的细菌、小鼠胚胎成纤维细胞（MEF）和人类成纤维细胞中利用这些测定。我们的第一个目标（具体目标 1 和 2）是鉴定能够将 MDS 转化为 DSB 的细菌和哺乳动物 DNA 修复酶。我们将使用模拟电离辐射损伤的定义 MDS 来评估细菌 AP 核酸内切酶、Fpg 和 Ogg1/Neil1 将 MDS 转化为 DSB 的能力。将检查包含 2 或 3 个病变的 MDS。我们假设细菌 AP 核酸内切酶将能够切割紧密相对的 AP 位点，即使在碱基附近存在损伤的情况下也是如此，这使它们成为使肿瘤细胞对癌症治疗敏感的主要候选者。我们已经确定，与大肠杆菌不同，哺乳动物细胞不容易将 MDS 转化为 DSB，并假设 NHEJ 是两种细胞类型中 MDS 生物学结果差异的原因。通过使用缺乏 NHEJ 蛋白的哺乳动物细胞和被操纵表达原核生物 NHEJ 系统的大肠杆菌，我们的第二个目标（目标 3）是测试 NHEJ 蛋白是否阻止 MDS 向 DSB 的转化。我们还将确定哪种 NHEJ 蛋白参与了这种 DSB 避免机制。总之，这项工作将提供设计更好的癌症治疗所需的基础知识。我们将通过基因治疗方法鉴定可在肿瘤细胞中过度表达的候选DNA修复酶，以增强电离辐射和化疗的杀伤力，并确定是否有必要禁用NHEJ以提高基因治疗的杀伤力。