Cellular Functions of Eukaryotic DNA Ligases

真核 DNA 连接酶的细胞功能

• 批准号: 10227946
• 负责人: Anthony J Davis
• 金额: $ 36.95万
• 依托单位: UNIVERSITY OF NEW MEXICO HEALTH SCIS CTR
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10227946
• 关键词: Adjuvant; Architecture; Binding; Biochemical; Biological; Biological Assay; Cancer cell line; Catalytic Domain; Cell Culture Techniques; Cell Cycle; Cell Death; Cell Survival; Cell physiology; Cells; Colorectal Cancer; Complex; DNA; DNA Double Strand Break; DNA Ligases; DNA Repair; DNA Repair Pathway; DNA ligase III; Dangerousness; Defect; Double Strand Break Repair; Effectiveness; Enzymes; FDA approved; Funding; G1 Phase; G2 Phase; Genes; Genetic; Genome Stability; Genomic Instability; Human; Incidence; Interphase Cell; Ionizing radiation; LIG4 gene; Laboratories; Lead; Libraries; Ligase; Ligation; Link; Maintenance; Malignant Neoplasms; Modeling; Monitor; Mus; Non-Malignant; Nonhomologous DNA End Joining; Oranges; Pathway interactions; Pharmaceutical Chemistry; Pharmaceutical Preparations; Play; Proteins; Radiation Tolerance; Radiation therapy; Radiation-Sensitizing Agents; Radiobiology; Radiosensitization; Reagent; Resistance; Role; S Phase; Site; Slide; Structure; Structure-Activity Relationship; Testing; WNT Signaling Pathway; Work; XRCC4 gene; Xenograft procedure; Yeasts; analog; base; cancer cell; design; drug development; experience; flexibility; inhibitor/antagonist; insight; novel; phenoxyacetic acid; predictive modeling; protein protein interaction; radiation resistance; radioresistant; recruit; release factor; repaired; screening; therapy resistant; tumor; tumor growth

PROJECT ABSTRACT The repair of DNA double strand breaks (DSB) is critical for cell survival and the maintenance of genome stability. Most DSBs in human cells are repaired the classic non-homologous end joining (NHEJ) pathway, although a homology-dependent pathway that operates predominantly in the S and G2 phases of the cell cycle also plays an important role in DSB repair. Inactivation of either one of these pathways results in with increased cancer incidence because of increased genome instability. Furthermore, they appear to be frequently inactivated or dysregulated in cancer cells with defects in the homology-dependent pathway conferring sensitivity to PARP inhibitors. In this competitive renewal application, we are continuing to focus on the DNA ligase IV (LigIV)- dependent classic NHEJ pathway. Although studies by the Tomkinson laboratory and many others have provided detailed mechanistic insights into the mammalian and yeast NHEJ pathways, the mechanisms by which both strands of DSB ends are joined and the core NHEJ factors are released from ligated DNA not been definitively elucidated. In Aim 1, employing a novel assay that enables us to simultaneously monitor ligation of both strands and the association of NHEJ factors with the DNA, we will test the hypotheses that the ligations of both strands is co- ordinated by two molecules of LigIV and the ring-shaped Ku heterodimer remains topologically linked to the DNA duplex following ligation. The activity of the classic NHEJ pathway is a major determinant of radiosensitivity. In the past funding period, we showed that some cancers, identified by elevated levels of PARP and DNA ligase III, have reduced classic NHEJ activity. There is also emerging evidence that the expression levels of LigIV correlate with radioresistance and that radioresistant cancers with activated Wnt signalling have elevated levels of LigIV. This prompted us to identify three selective LigIV inhibitors by screening a library composed predominantly of FDA approved drugs. In Aim 2, we will further characterize these inhibitors and determine structure-activity relationships. Active compounds will also be evaluated for LigIV- dependent activity in cell-based assays. In Aim 3, compounds with LigIV-dependent activity in cell-based assays will be further characterized and used as probes to determine the effect of inhibiting classic NHEJ in non-malignant and cancer cells. In addition, the ability of the LigIV inhibitors to enhance the efficacy of ionizing radiation in reducing tumor growth will be evaluated in mouse xenograft studies with radiosensitive and radioresistant cancer cell lines. We envision that the proposed studies will provide novel insights into the mechanism and contribution of LigIV- dependent NHEJ to DSB repair in non-malignant and cancer cells and will generate novel reagents to evaluate the utility of LigIV inhbitors in enhancing the efficacy and fidelity of gene editing and as radiosensitizers that increase the effectiveness of tumor-targeted radiation therapy.

项目摘要 DNA双链断裂（DSB）的修复对于细胞存活和维持至关重要 基因组稳定性。人类细胞中的大多数DSB修复了经典的非同源末端连接 （nhej）途径，尽管同源性途径主要在s中运行 细胞周期的G2阶段在DSB修复中也起着重要作用。两者的失活 由于基因组增加，这些途径之一导致癌症发病率增加 不稳定。此外，它们似乎在癌细胞中经常被灭活或失调 与同源依赖性途径中的缺陷赋予对PARP抑制剂的敏感性。在这个 竞争性更新应用，我们将继续专注于DNA连接酶IV（Ligiv） - 依赖的经典NHEJ途径。尽管汤姆金森实验室的研究和许多 其他人则对哺乳动物和酵母NHEJ提供了详细的机械见解 途径，连接DSB末端的两个链和核心NHEJ的机制 从连接的DNA中释放因子，未明确阐明。在AIM 1中，采用 使我们能够同时监测链和关联的新颖测定法 在使用DNA的NHEJ因子中，我们将测试这两个链的连接的假设是 由两个分子的联络分子和环形ku异二聚体围起来。 连接后与DNA双链体链接。经典NHEJ途径的活动是主要的 放射敏性的决定因素。在过去的资金期间，我们证明了一些癌症， 通过PARP和DNA连接酶III水平升高，已降低了经典NHEJ活性。 还有新的证据表明，恋的表达水平与放射线相关 并且具有活化的Wnt信号传导的辐射耐药癌具有升高的连接水平。这 促使我们通过筛选组成的库来识别三个选择性连接抑制剂 主要是FDA批准的药物。在AIM 2中，我们将进一步描述这些抑制剂，并 确定结构活性关系。还将评估活性化合物的连蛋白 基于细胞的测定中的依赖活性。在AIM 3中，具有连接活性的化合物 基于细胞的测定将进一步表征并用作探针来确定 抑制非恶性和癌细胞中的经典NHEJ。另外，联络的能力 将评估抑制剂以增强电离辐射在还原肿瘤生长中的疗效 在小鼠异种移植研究中，用于放射敏感和放射耐药的癌细胞系。我们设想 拟议的研究将提供有关联络机制和贡献的新见解。 依赖于非恶性和癌细胞中DSB修复的NHEJ，并将产生新颖的 试剂评估联络不稳定性在增强基因的功效和保真度方面的效用 编辑和作为提高受肿瘤辐射疗法有效性的放射增敏剂。

项目成果

Completion of base excision repair by mammalian DNA ligases.

• DOI: 10.1016/s0079-6603(01)68097-8
• 发表时间: 2001
• 期刊: Progress in nucleic acid research and molecular biology
• 作者: A. Tomkinson;Ling Chen;Zhiwan Dong;John B. Leppard;David S. Levin;Z. Mackey;Teresa A. Motycka

Biochemical and genetic characterization of the DNA ligase encoded by Saccharomyces cerevisiae open reading frame YOR005c, a homolog of mammalian DNA ligase IV.

酿酒酵母开放阅读框 YOR005c（哺乳动物 DNA 连接酶 IV 的同源物）编码的 DNA 连接酶的生化和遗传特征。

• DOI: 10.1093/nar/26.24.5676
• 发表时间: 1998
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Ramos,W;Liu,G;Giroux,CN;Tomkinson,AE
• 通讯作者: Tomkinson,AE

Mammalian DNA ligase III: molecular cloning, chromosomal localization, and expression in spermatocytes undergoing meiotic recombination.

哺乳动物 DNA 连接酶 III：分子克隆、染色体定位和进行减数分裂重组的精母细胞中的表达。

• DOI: 10.1128/mcb.15.10.5412
• 发表时间: 1995
• 期刊: Molecular and cellular biology
• 影响因子: 5.3
• 作者: Chen,J;Tomkinson,AE;Ramos,W;Mackey,ZB;Danehower,S;Walter,CA;Schultz,RA;Besterman,JM;Husain,I
• 通讯作者: Husain,I

Structure and function of mammalian DNA ligases.

• DOI: 10.1016/s0921-8777(97)00050-5
• 发表时间: 1998-02
• 期刊: Mutation research
• 作者: A. Tomkinson;Z. Mackey

Restoration of DNA repair mitigates genome instability and increases productivity of Chinese hamster ovary cells.

• DOI: 10.1002/bit.28016
• 发表时间: 2022-03
• 期刊: Biotechnology and bioengineering
• 影响因子: 3.8
• 作者: Spahn PN;Zhang X;Hu Q;Lu H;Hamaker NK;Hefzi H;Li S;Kuo CC;Huang Y;Lee JC;Davis AJ;Ly P;Lee KH;Lewis NE
• 通讯作者: Lewis NE