Chromosome instability resulting from double-strand breaks near telomeres

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（DSBs）高度敏感，在端粒附近用I-SceI内切酶诱导的DSBs比I-SceI诱导的其他位置的DSBs更容易导致大的缺失、总体染色体重排（GCRs）和染色体不稳定。重要的是，端粒附近的DSBs引起的重排与人类癌细胞中常见的重排相同，这使我们提出端粒附近的DSBs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。染色体愈合很少在其他位置的dsb上观察到，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。本研究将探讨端粒区对dsb的敏感性机制和染色体愈合的调节机制。这些研究将解决顺式作用的端粒蛋白直接抑制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细胞应答的ATM和MRE11蛋白的证据一致。在Aim 1A中，我们将通过确定哪些DNA修复蛋白与i - scii诱导的DSB共定位来表征端粒区域的DNA修复缺陷。这将涉及细胞系，其中DSB的位置用绿色荧光蛋白（GFP）标记，通过在I-SceI位点附近插入256个LacO操纵子拷贝，并表达与LacO操纵子结合的LacI-GFP融合蛋白。在Aim 1B中，我们将使用含有GFP基因和端粒附近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Aim 1C中，我们将使用包含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Aim 2A中，我们将使用一种新的实时定量PCR检测染色体愈合，以监测端粒蛋白或ATM的敲低如何影响染色体愈合的频率，这些细胞克隆是通过使用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Aim 2B中，我们将使用在Aim 1A中使用的含有标记有LacI-GFP融合蛋白的dsb的相同细胞克隆，比较在亚端粒和间质dsb中PIF1解解酶（一种已知抑制酵母染色体愈合的蛋白质）的外观。

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. PUBLIC HEALTH RELEVANCE: Chromosome instability is an important factor in promoting the multiple genetic changes leading to cancer. We have demonstrated that one mechanism for chromosome instability in human cancer cells involves the sudden loss of telomeres, the caps that protect the ends of chromosomes and prevent chromosome fusion. This proposal will investigate the mechanisms for two factors that we have shown are important in this process. The first is the sensitivity of regions near telomeres to double-strand breaks, which we believe is an important factor in the increased rate of telomere loss in cancer cells. The second is restoration of lost telomeres, called chromosome healing, which we have shown can prevent chromosome instability due to telomere loss. Understanding the mechanisms of telomere loss and chromosome healing in human cancer cells may lead to new approaches for the limiting the chromosome instability responsible for cancer cell progression and resistance to anti-cancer therapies.