"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）：由治疗和环境电离辐射（IR）/放射性药物引起的基因组损伤簇包括具有不可粘末端的双链断裂（DSB），以及更丰富的基础/糖氧化产物群，Abasic（AP）位点和单链（SSSTRAND BREAKS）。高度细胞毒性的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，这些基因组在所有细胞中通过非同源末端连接（NHEJ）在S/G2细胞中通过非误差端连接（NHEJ）进行。替代末端连接（Alt-EJ）还维修DSB，包括通过DNA糖基酶（DGS），AP-核酸酶（APE1）和其他通过BER/SSBR途径修复双链非DSB病变簇的DSB。 NHEJ启动KU抑制使用基于微学的SSBR的Alt-Ej比NHEJ更容易发生。 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.该项目的中心假设是，NHEJ先于由KU（KU70/80）协调的Alt-EJ/BER，该Alt-EJ/BER在DSB处招募了DNA-PKC，然后由DSB的终端处理和DNA Ligase4/XRCC4/XRCC4/XLF进行了DNA Ligase4/XRC。基于我们的初步研究，表明：（a）DG/APE1免疫复合物（ICS）中存在的KU抑制它们； (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使用SSBR蛋白和一组不同的最终加工酶，Alt-Ej/ber。我们将通过追求三个目标来检验这一全面假设的各个方面：目标1。评估Alt-Ej对放射性抗抑郁剂的贡献及其在使用记者质粒质粒分析和体外与细胞基因组修复的分析相似，并显示Alt-Ej/Ber-ej/Ber-ej/berej nhhej in hadiejection nhhej in gropotection and-ecloter plastId分析的贡献及其在修复辐射损害方面的需求。目的2。测试HNRNP-U和KU通过DNA-PK介导的磷酸化协调NHEJ和Alt-EJ的假设。目的3。为了测试以下假设：NHEJ和Alt-EJ/BER蛋白会形成能力竞争力的动态复合物，该假设是由辐射/Enediyne药物调节的动态复合物，涉及与KU的物理相互作用。我们将表征Alt-EJ的末端加工酶和间隙填充DNA聚合酶（S）。这些研究将深刻增强我们对复杂辐射诱导的基因组损伤的修复的理解，以及Alt-EJ对肿瘤辐射的贡献，并有助于鉴定出新的治疗靶标，例如KU，例如肿瘤的同时放射性化和正常细胞的放射性抑制作用。