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：
8849386
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 2017-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8849386
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 Cre-LoxP DNA Double Strand Break DNA Repair 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 endodeoxyribonuclease SceI gene repair healing helicase interstitial knock-down novel novel strategies prevent promoter repaired response restoration telomere telomere loss

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（由申请人提供）：端粒在保护染色体的末端和防止染色体融合方面起着重要作用。我们已经证明，端粒附近的区域对DNA双链断裂（DSB）高度敏感，因为端粒附近的I-SCEI核酸内切酶诱导的DSB更有可能导致大量缺失，总染色体重排（GCRS），以及比I-SCEI-ISBS诱导的DSBS在其他位置上。重要的是，由端粒附近的DSB引起的重排是人类癌细胞中常见的重排，这使我们提出端粒附近的DSB是致癌作用的重要机制。我们还表明，通过在DSB的位点添加新的端粒（称为染色体愈合的过程），可以通过在端粒附近引起的DSB引起的染色体不稳定性。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由于端粒附近的DSB引起的染色体不稳定性的重要机制。该建议将研究负责端粒区域对DSB敏感性的机制以及染色体愈合的调节机制。这些研究将解决以下假设：顺式作用端粒蛋白直接抑制DSB修复并促进染色体愈合，这与端粒蛋白TRF2抑制了参与细胞对DSB的细胞反应的ATM和MRE11蛋白的证据一致。在AIM 1A中，我们将通过确定哪种DNA修复蛋白与I-SCEI诱导的DSB共定位，来表征端粒区域中的DNA修复缺陷。这将涉及细胞系，其中通过插入与I-SCEI位点相邻的LACO操纵子的256份副本以及LACI-GFP融合蛋白的表达，其中DSB的位置用绿色荧光蛋白（GFP）标记，并结合LACO操纵子。在AIM 1B中，我们将使用含有GFP基因的细胞克隆和与端粒相邻的I-SCEI位点来监控端粒蛋白或DSB修复蛋白的敲低如何影响大删除的频率。在AIM 1C中，我们将使用含有由核体内重排激活的GFP基因的细胞克隆以及由染色体内重排激活的GFP基因的细胞克隆以及由染色体界面部重排激素激活的DSRED基因的DSB修复蛋白。在AIM 2A中，我们将使用一种新型的实时定量PCR分析进行染色体愈合，以监测端粒蛋白或ATM的敲低如何影响通过将端粒移动到使用CRE/LoxpP PPP介导的重新组成的ISEGENIC细胞克隆中的染色体愈合频率。在AIM 2B中，我们将比较PIF1解旋酶的外观，PIF1解旋酶是一种已知的蛋白质，可在酵母中，亚电体和间质DSB中抑制染色体愈合，使用和不敲除TRF2或ATM的情况下，使用与LACI-GFP融合蛋白在AIM 1A中使用的laci-GFP融合蛋白标记的同一细胞克隆。