"Repair Co-ordination of Radiation-Induced Clustered Damage In Mammalian Genomes"

“哺乳动物基因组中辐射诱导的聚集性损伤的修复协调”

• 批准号: 9010941
• 负责人: Sankar Mitra
• 金额: $ 33.1万
• 依托单位: METHODIST HOSPITAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-05 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9010941
• 关键词: ATM activation; Binding; Biological Assay; Cell Cycle Checkpoint; Cell Survival; Cells; Comet Assay; Complex; DNA; DNA Ligases; DNA Ligation; DNA Repair Pathway; DNA glycosylase; DNA-(apurinic or apyrimidinic site) lyase; DNA-Directed DNA Polymerase; DNA-PKcs; DNA-dependent protein kinase; Data; Diagnostic Procedure; Double Strand Break Repair; Environment; Enzymes; Excision; G22P1 gene; Genome; Genomics; Goals; Health; Heterogeneous-Nuclear Ribonucleoprotein U; In Vitro; Ionizing radiation; Joints; Kinetics; Lead; Lesion; Ligation; Mammals; Maps; Mediating; Modification; Molecular; Normal Cell; Nuclear Extract; Nucleotides; PARP inhibition; Pathway interactions; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Plasmids; Polymerase; Process; Proteins; Protocols documentation; Radiation; Radiation Accidents; Radiation Tolerance; Radioprotection; Radioresistance; Radiosensitization; Recruitment Activity; Repair Complex; Reporter; Resources; Signal Transduction; Single Strand Break Repair; Site; Testing; Therapeutic; Therapeutic Intervention; Tissues; Toxic effect; Translations; XRCC1 gene; XRCC4 gene; XRCC5 gene; base; cytotoxic; experience; genome integrity; homologous recombination; improved; in vivo; innovation; irradiation; mammalian genome; mutant; neoplastic cell; new therapeutic target; novel; novel strategies; oxidation; prevent; repaired; seal; space travel; sugar; tumor

DESCRIPTION (provided by applicant): Clusters of genomic damage induced by therapeutic and environmental ionizing radiation (IR)/radiomimetic drugs include double-strand breaks (DSBs) with nonligatable ends, and more abundant clusters of base/sugar oxidation products, abasic (AP) sites and single-strand breaks (SSBs). Highly cytotoxic DSBs, also formed during SSB replication, activate cell-cycle checkpoints and promote DSB repair (DSBR), which in mammalian genomes occurs via nonhomologous end joining (NHEJ) in all cells, and by error-free homologous recombination (HR) in S/G2 cells. Alternative end joining (Alt-EJ) also repairs DSBs, including those generated during the repair of bi-stranded non-DSB lesion clusters by DNA glycosylases (DGs), AP-endonuclease (APE1), and others via the BER/SSBR pathway. NHEJ-initiating Ku inhibits Alt-EJ which using microhomology- based SSBR is more error prone than NHEJ. Radiosensitivity caused by DG/APE1 deficiency and of HR negative tumors by inhibition of PARP-1-initiated SSBR indicates Alt-EJ/SSBR's significant contribution to radioresistance, which must be coordinated with NHEJ, the predominant DSBR pathway in mammals.BER prior to NHEJ would cause secondary DSBs which near preexisting DSBs would lead to larger deletions. This project's central hypothesis is that NHEJ precedes Alt-EJ/BER, coordinated by Ku (Ku70/80), which recruits DNA-PKcs at the DSB, followed by the DSB's end processing and re-ligation by DNA ligase4/XRCC4/XLF. Based on our preliminary studies showing that: (a) Ku present in DG/APE1 immunocomplexes (ICs) inhibits them; (b) the Ku IC from irradiated cells performs NHEJ of a novel linearized plasmid substrate with dirty ends, whose in-cell repair involves both NHEJ and Alt-EJ, we hypothesize that hnRNP-U, present in Ku IC only after irradiation, and phosphorylated by DNA-PK during NHEJ, relieves Ku inhibition, thus acting as a molecular switch for transition to Alt-EJ/BER which utilizes SSBR proteins and a distinct set of end-processing enzymes. We will test various facets of this comprehensive hypothesis by pursuing three aims: Aim 1. To assess the contribution of Alt-EJ to radioresistance and its requirements in repairing radiation damage using reporter plasmid assays in-cell and in vitro, in parallel with analysis of cell genome repair, and to show that Alt-EJ/BER is additive to NHEJ in radioprotection. Aim 2. To test the hypothesis that hnRNP-U and Ku together coordinate NHEJ and Alt-EJ via DNA-PK-mediated phosphorylation. Aim 3. To test the hypothesis that NHEJ and Alt-EJ/BER proteins form repair-competent, dynamic complexes modulated by radiation/enediyne drugs involving physical interaction with Ku. We will characterize the end- processing enzymes and gap-filling DNA polymerase(s) for Alt-EJ. These studies will profoundly enhance our understanding of the repair of complex radiation-induced genomic damage, and of the contribution of Alt-EJ to radioresistance of tumors, and help identify novel therapeutic targets such as Ku for simultaneous radiosensitization of tumors and radioprotection of normal cells.

描述（由申请人提供）：治疗和环境电离辐射（IR）/拟放射药物诱导的基因组损伤簇包括具有不可连接末端的双链断裂（DSB），以及更丰富的碱基/糖氧化产物簇，无碱基（AP） ）位点和单链断裂（SSB）。高细胞毒性 DSB 也在 SSB 复制过程中形成，激活细胞周期检查点并促进 DSB 修复 (DSBR)，在哺乳动物基因组中，DSB 修复通过所有细胞中的非同源末端连接 (NHEJ) 以及通过无错误同源重组 (HR) 发生。 S/G2 细胞。替代末端连接 (Alt-EJ) 还可修复 DSB，包括 DNA 糖基酶 (DG)、AP 核酸内切酶 (APE1) 和其他通过 BER/SSBR 途径修复双链非 DSB 损伤簇期间产生的 DSB。 NHEJ 启动 Ku 抑制 Alt-EJ，而使用基于微同源性的 SSBR 比 NHEJ 更容易出错。 DG/APE1 缺陷引起的放射敏感性和通过抑制 PARP-1 启动的 SSBR 引起的 HR 阴性肿瘤表明 Alt-EJ/SSBR 对放射抗性的显着贡献，这必须与 NHEJ（哺乳动物中主要的 DSBR 途径）协调。BER 优先于 NHEJ会导致次级 DSB 靠近先前存在的 DSB，从而导致更大的删除。该项目的中心假设是NHEJ先于Alt-EJ/BER，由Ku (Ku70/80)协调，在DSB处招募DNA-PKc，随后是DSB的末端处理并通过DNA连接酶4/XRCC4/XLF重新连接。根据我们的初步研究表明： (a) DG/APE1 免疫复合物 (IC) 中存在的 Ku 会抑制它们； （b）来自受辐射细胞的Ku IC执行具有脏末端的新型线性化质粒底物的NHEJ，其细胞内修复涉及NHEJ和Alt-EJ，我们假设hnRNP-U仅在辐射后才存在于Ku IC中，并且NHEJ 期间被 DNA-PK 磷酸化，解除 Ku 抑制，从而充当过渡到 Alt-EJ/BER 的分子开关，后者利用 SSBR 蛋白和一组独特的末端加工酶。我们将通过追求三个目标来测试这一综合假设的各个方面： 目标 1. 使用细胞内和体外报告质粒测定来评估 Alt-EJ 对放射抗性的贡献及其修复辐射损伤的要求，同时分析细胞基因组修复，并证明 Alt-EJ/BER 在辐射防护方面对 NHEJ 具有补充作用。目标 2. 检验 hnRNP-U 和 Ku 通过 DNA-PK 介导的磷酸化共同协调 NHEJ 和 Alt-EJ 的假设。目标 3. 检验 NHEJ 和 Alt-EJ/BER 蛋白形成具有修复能力的动态复合物的假设，该复合物由辐射/烯二炔药物调节，涉及与 Ku 的物理相互作用。我们将表征 Alt-EJ 的末端加工酶和缺口填充 DNA 聚合酶。这些研究将深刻增强我们对复杂辐射诱导基因组损伤的修复以及 Alt-EJ 对肿瘤放射抗性的贡献的理解，并有助于确定新的治疗靶点，例如同时对肿瘤进行放射增敏和对正常细胞进行放射防护的 Ku 。