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

描述（由申请人提供）：治疗和环境电离辐射（IR）/拟放射性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、脱碱基（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），这在哺乳动物基因组中通过所有细胞中的非同源末端连接（NHEJ）和S/G2细胞中的无错误同源重组（HR）发生。选择性末端连接（Alt-EJ）也修复DSB，包括通过DNA糖基化酶（DG）、AP内切核酸酶（APE 1）和其他经BER/SSBR途径修复双链非DSB损伤簇期间产生的DSB。NHEJ起始Ku抑制Alt-EJ，Alt-EJ使用基于微同源性的SSBR比NHEJ更容易出错。DG/APE 1缺陷引起的放射敏感性以及通过抑制PARP-1引发的SSBR而引起的HR阴性肿瘤的放射敏感性表明Alt-EJ/SSBR对放射抵抗性有显着贡献，这必须与哺乳动物中主要的DSBR途径NHEJ协调。NHEJ之前的BER会导致继发性DSB，这些DSB靠近先前存在的DSB将导致更大的缺失。该项目的中心假设是NHEJ先于Alt-EJ/BER，由Ku（Ku 70/80）协调，其在DSB处招募DNA-PKcs，随后是DSB的末端加工和通过DNA连接酶4/XRCC 4/XLF的重新连接。我们的初步研究表明：（a）存在于DG/APE 1免疫复合物（IC）中的Ku抑制了它们;（B）来自辐射细胞的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两者，我们假设仅在辐射后存在于Ku IC中并且在NHEJ期间被DNA-PK磷酸化的hnRNP-U解除Ku抑制，因此作为一个分子开关，用于转换到Alt-EJ/BER，其利用SSBR蛋白和一组不同的末端加工酶。我们将通过追求三个目标来测试这个综合假设的各个方面：目标1。评估Alt-EJ对辐射抗性的贡献及其在修复辐射损伤中的要求，使用细胞内和体外报告质粒测定，与细胞基因组修复分析平行，并显示Alt-EJ/BER在辐射防护中与NHEJ相加。目标二。为了检验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：为了检验NHEJ和Alt-EJ/BER蛋白形成由涉及与Ku的物理相互作用的辐射/烯二炔药物调节的具有修复能力的动态复合物的假设。我们将表征Alt-EJ的末端加工酶和间隙填充DNA聚合酶。这些研究将深刻地提高我们对复杂辐射诱导的基因组损伤的修复以及Alt-EJ对肿瘤辐射抗性的贡献的理解，并有助于确定新的治疗靶点，如Ku，用于同时对肿瘤进行放射增敏和对正常细胞进行放射保护。

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.