DNA Repair of Multiply Damaged Sites in Cells

描述（由申请方提供）：某些化疗和放疗在细胞中产生由DMA碱基损伤和链断裂组成的多重损伤位点（MDS）。由电离辐射产生的MDS可以由< 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），但是人类细胞中的非同源末端连接（NHEJ）起到防止修复中间体转化为致死事件的作用。我们已经开发了将合成MDS置于质粒的荧光素酶编码区内的测定。将质粒转移到细胞中，报告活性的丧失、质粒的破坏或缺失的引入表明DSB形成。我们将在细菌、小鼠胚胎成纤维细胞（MEF）和人成纤维细胞中利用这些测定法，这些成纤维细胞在碱基切除修复（BER）或非同源末端连接（NHEJ）中是熟练的和缺乏的。我们的第一个目标（具体目标1和2）是鉴定可以将MDS转化为DSB的细菌和哺乳动物DNA修复酶。我们将使用定义的MDS模拟电离辐射损伤，以评估细菌AP核酸内切酶Fpg和Ogg 1/Neil 1将MDS转化为DSB的能力。将检查包含2或3处病变的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，即使在存在附近碱基损伤的情况下，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NHEJ蛋白缺陷的哺乳动物细胞和被操纵以表达原核生物NHEJ系统的大肠杆菌，我们的第二个目标（目的3）是测试NHEJ蛋白是否阻止MDS转化为DSB。我们还将确定哪种NHEJ蛋白参与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本知识。我们将确定候选DNA修复酶，可以在肿瘤细胞中过度表达，使用基因治疗方法，以提高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。

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.