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发挥着重要作用。我们已经证明了端粒附近的区域对DNA双链断裂(DSB)高度敏感，因为在端粒附近用I-SCEI内切酶诱导的DSB比I-SCEI在其他位置诱导的DSB更有可能导致大的缺失、总的染色体重排(GCRs)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，这导致我们提出端粒附近的DSB是癌症发生的重要机制。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处增加一个新的端粒来防止，这一过程称为染色体修复。染色体修复在其他位置的DSB很少观察到，因此我们提出染色体修复是防止端粒附近DSB引起的染色体不稳定的重要机制。这项建议将探讨端粒区域对DSB敏感的机制以及染色体愈合的调节机制。这些研究将解决这样的假设，即顺式作用的端粒蛋白直接抑制DSB修复并促进染色体愈合，这与端粒蛋白TRF2抑制参与细胞对DSB的反应的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。这将涉及到这样的细胞系，在其中DSB的位置用绿色荧光蛋白(GFP)标记，方法是在I-SCEI位点附近插入256个拷贝的LACO操纵子，并表达与LACO操纵子结合的LacI-GFP融合蛋白。在目标1B中，我们将使用包含GFP基因和靠近端粒的I-SCEI位点的细胞克隆来监测端粒蛋白或DSB修复蛋白的敲除如何影响大片段缺失的频率。在AIM 1C中，我们将使用包含染色体内重排激活的GFP基因和染色体间重排激活的DsRed基因的细胞克隆来鉴定参与染色体异常形成的DSB修复蛋白。在Aim 2A中，我们将使用一种新的用于染色体修复的实时定量PCR方法来监控端粒蛋白或ATM的敲除如何影响同基因细胞克隆中染色体修复的频率，这些克隆是通过使用Cre/loxP介导的重组将端粒移动到I-SCEI位点附近而产生的。在目标2B中，我们将使用与目标1A中使用的标记有LacI-GFP融合蛋白的DSB相同的细胞克隆，比较PIF1解旋酶(一种已知在酵母中抑制染色体愈合的蛋白质)在TRF2或ATM被敲除和不被击倒的情况下在亚端粒和间质DSB上的外观。 公共卫生相关性：染色体不稳定是促进导致癌症的多种基因变化的重要因素。我们已经证明，人类癌细胞中染色体不稳定的一个机制涉及端粒的突然丢失，端粒是保护染色体末端并阻止染色体融合的帽子。这项提案将调查两个因素的机制，我们已经证明这两个因素在这一进程中很重要。首先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端粒损失率增加的一个重要因素。第二种是修复丢失的端粒，称为染色体修复，我们已经证明这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修复的机制可能会为限制导致癌细胞进展和抗癌治疗耐药的染色体不稳定性提供新的途径。

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.