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Chromosome instability resulting from double-strand breaks near telomeres

端粒附近双链断裂导致染色体不稳定

基本信息

批准号：
8323913
负责人：
John P. Murnane
金额：
$ 23.97万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-07-06 至 2016-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8323913
关键词：
Address Affect Appearance Binding Biological Assay Cell Line Cells Chimeric Proteins Chromosomal Breaks Chromosomal Instability Chromosomal Rearrangement Chromosome abnormality Chromosomes Clone Cells Code DNA Double Strand Break DNA Repair DNA Sequence Rearrangement DNA repair protein Defect Double Strand Break Repair DsRed Frequencies Genes Genetic Recombination Green Fluorescent Proteins Healed Human Knock-out Label Lead Location Malignant Neoplasms Mammalian Cell Mediating Modification Monitor Mutation Nonhomologous DNA End Joining Operon Phosphorylation Plasmids Play Process Protein Binding Proteins Regulation Resistance Role Site System Telomere Capping Telomeric Repeat Binding Protein 2 Time Yeasts cancer cell cancer therapy carcinogenesis chromosome loss endodeoxyribonuclease SceI gene repair healing helicase interstitial knock-down novel novel strategies prevent promoter repaired response restoration telomere

项目摘要

DESCRIPTION (provided by applicant): Telomeres play an important role in protecting the ends of chromosomes and preventing chromosome fusion. We have demonstrated that the regions near telomeres are highly sensitive to DNA double-strand breaks (DSBs), in that DSBs induced with I-SceI endonuclease near telomeres are much more likely to result in large deletions, gross chromosome rearrangements (GCRs), and chromosome instability than I-SceI-induced DSBs at other locations. Importantly, the rearrangements caused by DSBs near telomeres are the same rearrangements commonly found in human cancer cells, leading us to propose that DSBs near telomeres are an important mechanism in carcinogenesis. We have also shown that the chromosome instability caused by DSBs near telomeres can be prevented by the addition of a new telomere at the site of the DSB, a process called chromosome healing. Chromosome healing is rarely observed at DSBs at other locations, and therefore we have proposed that chromosome healing is an important mechanism for preventing chromosome instability due to DSBs near telomeres. This proposal will investigate the mechanism responsible for the sensitivity of telomeric regions to DSBs and the mechanism of regulation of chromosome healing. These studies will address the hypothesis that cis-acting telomeric proteins directly inhibit DSB repair and promote chromosome healing, consistent with evidence that the telomeric protein TRF2 inhibits the ATM and MRE11 proteins involved in the cellular response to DSBs. In Aim 1A we will characterize the DNA repair defect in telomeric regions by determining which DNA repair proteins co-localize with the I-SceI-induced DSB. This will involve cell lines in which the location of the DSB is marked with green fluorescent protein (GFP) by inserting 256 copies of a LacO operon adjacent to the I-SceI site, and expression of LacI-GFP fusion protein, which binds the LacO operon. In Aim 1B we will use cell clones containing a GFP gene and an I-SceI site adjacent to a telomere to monitor how knockdown of telomeric proteins or DSB repair proteins affects the frequency of large deletions. In Aim 1C we will characterize the DSB repair proteins involved in the formation of chromosome aberrations using cell clones that contain a GFP gene activated by intrachromosomal rearrangements, and a DsRed gene activated by interchromosomal rearrangemetns. In Aim 2A we will use a novel real-time quantitative PCR assay for chromosome healing to monitor how knockdown of telomeric proteins or ATM affects the frequency of chromosome healing in isogenic cell clones generated by moving a telomere to a location adjacent to the I-SceI site using Cre/LoxP-mediated recombination. In Aim 2B we will compare the appearance of PIF1 helicase, a protein known to inhibit chromosome healing in yeast, at subtelomeric and interstitial DSBs with and without knockdown of TRF2 or ATM, using the same cell clones containing the DSBs marked with the LacI-GFP fusion protein used in Aim 1A.

描述（申请人提供）：端粒在保护染色体末端和防止染色体融合方面发挥着重要作用。我们已经证明，端粒附近的区域对 DNA 双链断裂 (DSB) 高度敏感，因为端粒附近 I-SceI 核酸内切酶诱导的 DSB 比其他位置的 I-SceI 诱导的 DSB 更有可能导致大缺失、总染色体重排 (GCR) 和染色体不稳定。重要的是，端粒附近的 DSB 引起的重排与人类癌细胞中常见的重排相同，因此我们提出端粒附近的 DSB 是致癌的重要机制。我们还表明，端粒附近的 DSB 引起的染色体不稳定可以通过在 DSB 位置添加新的端粒来预防，这一过程称为染色体愈合。在其他位置的 DSB 上很少观察到染色体愈合，因此我们提出染色体愈合是防止端粒附近 DSB 引起的染色体不稳定的重要机制。该提案将研究端粒区域对 DSB 的敏感性的机制以及染色体愈合的调节机制。这些研究将提出顺式作用端粒蛋白直接抑制 DSB 修复并促进染色体愈合的假设，这与端粒蛋白 TRF2 抑制参与 DSB 细胞反应的 ATM 和 MRE11 蛋白的证据一致。在目标 1A 中，我们将通过确定哪些 DNA 修复蛋白与 I-SceI 诱导的 DSB 共定位来表征端粒区域的 DNA 修复缺陷。这将涉及通过在 I-SceI 位点附近插入 256 个 LacO 操纵子拷贝，用绿色荧光蛋白 (GFP) 标记 DSB 位置的细胞系，并表达与 LacO 操纵子结合的 LacI-GFP 融合蛋白。在目标 1B 中，我们将使用含有 GFP 基因和邻近端粒的 I-SceI 位点的细胞克隆来监测端粒蛋白或 DSB 修复蛋白的敲低如何影响大缺失的频率。在目标 1C 中，我们将使用含有由染色体内重排激活的 GFP 基因和由染色体间重排激活的 DsRed 基因的细胞克隆来表征参与染色体畸变形成的 DSB 修复蛋白。在目标 2A 中，我们将使用一种新型的染色体愈合实时定量 PCR 测定来监测端粒蛋白或 ATM 的敲低如何影响同基因细胞克隆中染色体愈合的频率，这些克隆是通过使用 Cre/LoxP 介导的重组将端粒移动到 I-SceI 位点附近的位置而产生的。在目标 2B 中，我们将使用含有目标 1A 中使用的 LacI-GFP 融合蛋白标记的 DSB 的相同细胞克隆，比较 PIF1 解旋酶（一种已知抑制酵母染色体愈合的蛋白质）在敲除 TRF2 或 ATM 和未敲除 TRF2 或 ATM 的情况下在亚端粒和间质 DSB 中的外观。