Chromosome instability resulting from double-strand breaks near telomeres

描述（由申请人提供）：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内切酶诱导的DSB比其他位置的I-SceI诱导的DSB更可能导致大的缺失、总染色体重排（GCR）和染色体不稳定性。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，这使我们提出端粒附近的DSB是致癌的重要机制。我们还表明，由端粒附近的DSB引起的染色体不稳定性可以通过在DSB位点添加新的端粒来防止，这一过程称为染色体愈合。在其他位置的DSB中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。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控机制。这些研究将解决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TRF 2抑制参与细胞对DSB反应的ATM和MRE 11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。这将涉及这样的细胞系，其中通过在I-SceI位点附近插入256个拷贝的LacO操纵子，用绿色荧光蛋白（GFP）标记DSB的位置，并表达结合LacO操纵子的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在同基因细胞克隆通过移动端粒的位置附近的I-SceI网站使用Cre/LoxP介导的重组。在Aim 2B中，我们将比较PIF 1解旋酶（一种已知抑制酵母中染色体愈合的蛋白质）在TRF 2或ATM敲减和未敲减的亚端粒和间质DSB处的外观，使用含有Aim 1A中使用的LacI-GFP融合蛋白标记的DSB的相同细胞克隆。 公共卫生相关性：染色体不稳定性是促进导致癌症的多种遗传变化的重要因素。我们已经证明，人类癌细胞中染色体不稳定的一种机制涉及端粒的突然丢失，端粒是保护染色体末端并防止染色体融合的帽。本提案将研究我们已经证明在这一过程中很重要的两个因素的机制。第一个是端粒附近区域对双链断裂的敏感性，我们认为这是癌细胞中端粒丢失率增加的重要因素。第二个是恢复丢失的端粒，称为染色体愈合，我们已经证明可以防止由于端粒丢失而导致的染色体不稳定。了解人类癌细胞中端粒丢失和染色体愈合的机制可能会导致限制染色体不稳定性的新方法，这些不稳定性是癌细胞进展和对抗癌疗法的抗性的原因。

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.