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

描述(申请人提供)：治疗和环境电离辐射(IR)/仿射药物引起的基因组损伤包括末端不可连接的双链断裂(DSB)，以及更丰富的碱基/糖氧化产物簇、碱性(AP)位点和单链断裂(SSB)。在哺乳动物基因组中，双链断裂在所有细胞中通过非同源末端连接(NHEJ)和在S/G2细胞中无错误的同源重组(HR)发生。替代末端连接(Alt-EJ)也可以修复DSB，包括那些在DNA糖基酶(DGs)、AP-内切酶(APE1)和其他通过BER/SSBR途径修复双链非DSB病变簇过程中产生的DSB。NHEJ-发起Ku抑制Alt-EJ，使用基于微同源的SSBR比NHEJ更容易出错。DG/APE1缺乏引起的放射敏感性和PARP-1启动的SSBR抑制导致的HR阴性肿瘤表明Alt-EJ/SSBR在辐射抵抗中做出了重要贡献，这必须与哺乳动物中主要的DSBR途径NHEJ相协调。该项目的中心假设是，NHEJ先于Alt-EJ/BER，由Ku(Ku70/80)协调，在DSB招募DNA-PKcs，随后是DSB的末端加工和DNA连接酶4/XRCC4/XLF的重新连接。基于我们的初步研究表明：(A)存在于DG/APE1免疫复合物(IC)中的Ku抑制它们；(B)来自辐照细胞的Ku IC执行一种新型线性化的带有脏端的质粒底物的NHEJ，其细胞内修复涉及NHEJ和Alt-EJ，我们假设hnRNP-U仅在照射后存在于Ku IC中，并在NHEJ期间被DNA-PK磷酸化，从而作为向Alt-EJ/BER过渡的分子开关，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检验这一综合假说的各个方面：目的1.通过细胞内和体外报告质粒检测，结合细胞基因组修复分析，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，并证明Alt-EJ/BER在辐射防护方面是NHEJ的补充。目的2.验证hnRNP-U和Ku通过DNA-PK介导的磷酸化协同NHEJ和Alt-EJ的假说。目的3.验证NHEJ和Alt-EJ/BER蛋白在辐射/烯二炔类药物调控下与Ku形成具有修复能力的动态复合体的假设。我们将对ALT-EJ的末端加工酶和缺口填充DNA聚合酶(S)进行鉴定。这些研究将深刻地加深我们对复杂的辐射诱导的基因组损伤的修复以及Alt-EJ在肿瘤放射抵抗中的贡献的理解，并有助于寻找新的治疗靶点，如Ku，用于同时对肿瘤进行放射增敏和对正常细胞进行辐射防护。

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.