Roles of Telomeric Oxidative DNA Lesions in Telomere Length Regulation

该项目的目标是确定氧化DNA损伤如何调节同源基因导向的修复。 (HDR)通过交替延长端粒来维持端粒的通路(ALT) 机械装置。氧化还原调节功能障碍在癌症中很常见，并会升高活性氧 产生DNA损伤的物种。端粒对氧化损伤非常敏感，但却是必不可少的。 基因组的稳定性和持续的细胞增殖。功能失调的端粒阻止细胞分裂或驱动过度 癌前细胞中的染色体不稳定，可能导致细胞死亡。为了生存和实现无限 在增殖过程中，癌细胞通过激活端粒酶或丙氨酸氨基转移酶延长和稳定端粒。尽管数量较少 常见的ALT驱动的癌症是高度侵袭性的，预后很差。端粒单链突起 是端粒酶和ALT介导的端粒延长的底物，但可以自我折叠成稳定的 二次结构。此前，我们发现低水平的8oxoG通过改变结构来刺激端粒酶 和突出的可及性，但超过8oxoG的损伤会损害端粒复制。在这个项目中，我们将 验证8oxoG在端粒的形成和处理通过促进复制来调节ALT的假设 分叉停滞和改变端粒结构。使用创新的目标工具选择性地在以下位置诱导8oxoG 端粒，我们获得了初步数据，8oxoG增加了许多ALT标志，包括C-环， ALT相关的PML小体、端粒姐妹染色单体交换和有丝分裂DNA合成。目标1将 确定氧化碱基损伤如何调节人类癌细胞系的ALT和HDR活性 而且缺乏修缮能力。我们将在ALT和ALT中诱导端粒特异性损伤或普遍的氧化应激 端粒酶阳性细胞，并将测量细胞存活率、ALT表型和各种端粒参数。 ALT需要RAD51或RAD52，受多种端粒结构调控。Aim 2将研究如何 氧化损伤调节端粒RNA(Terra)的入侵和端粒R环的形成。使用 单分子荧光共振能量转移检测系统，我们在端粒中发现了8oxoG 双工促进了Terra关联。我们将使用互补的细胞研究来检查Terra 氧化损伤后重新聚集到端粒。目标3将研究端粒中的氧化损伤如何 悬垂通过RAD51介导链侵袭或RAD52介导链调节D-环的形成 退火法，通过互补性的单分子和生化实验。我们将进行蜂窝 研究确定RAD51或RAD52缺乏如何影响8oxoG诱导的ALT表型。这个项目 将填补我们对一般氧化应激，特别是8oxoG如何改变的理解中的一个重大空白 通过同源定向修复和ALT修复端粒。最终，这些知识将是非常有价值的 为了开发新的策略，1)保护端粒以减轻氧化应激对健康的影响 细胞或相反，2)抑制恶性ALT肿瘤的端粒修复以阻止增殖。

The goals of this project are to determine how oxidative DNA damage regulates the homology-directed repair (HDR) pathways that enable telomere maintenance by the alternative lengthening of telomeres (ALT) mechanisms. Dysfunctional redox regulation is common among cancers and elevates reactive oxygen species, which generate DNA lesions. Telomeres are highly susceptible to oxidative damage but are essential for genome stability and sustained cell proliferation. Dysfunctional telomeres arrest cell division or drive excess chromosomal instability in pre-malignant cells that can cause cell death. To survive and achieve unlimited proliferation, cancer cells elongate and stabilize telomeres by activating telomerase or ALT. Although less common, ALT-driven cancers are highly aggressive with poor prognosis. Telomeric single-stranded overhangs are substrates for both telomerase and ALT-mediated telomere elongation but can self-fold into stable secondary structures. Previously, we found that low 8oxoG levels stimulate telomerase by altering structure and overhang accessibility, but that excess 8oxoG damage impairs telomere replication. In this project we will test the hypothesis that 8oxoG formation and processing at telomeres modulate ALT by promoting replication fork stalling and altering telomere structures. Using an innovative targeting tool to selectively induce 8oxoG at telomeres, we obtained preliminary data that 8oxoG increases numerous ALT hallmarks including C-circles, ALT-associated PML bodies, and telomeric sister chromatid exchanges and mitotic DNA synthesis. Aim 1 will determine how oxidative base damage modulates ALT and HDR activity in human cancer cell lines, proficient and deficient for repair. We will induce telomere specific damage or general oxidative stress in ALT and telomerase positive cells, and will measure cell survival, ALT phenotypes, and various telomere parameters. ALT requires RAD51 or RAD52 and is regulated by a variety of telomeric structures. Aim 2 will examine how oxidative lesions modulate telomeric RNA (TERRA) invasion and R-loop formation into telomeres. Using a single molecule fluorescence resonance energy transfer detection system, we discovered 8oxoG in telomeric duplex facilitates TERRA association. We will use complementary cellular studies to examine TERRA recruitment to telomeres after oxidative damage. Aim 3 will examine how oxidative damage in the telomere overhang modulates D-loop formation by RAD51-mediated strand invasion, or RAD52-mediated strand annealing, by using complementary single molecule and biochemical experiments. We will conduct cellular studies to determine how RAD51 or RAD52 deficiency influence 8oxoG-induced ALT phenotypes. This project will fill a significant void in our understanding of how general oxidative stress and 8oxoG, in particular, alter telomere restoration by homology-directed repair and ALT. Ultimately, this knowledge will be highly valuable for developing new strategies that 1) preserve telomeres to mitigate the effects of oxidative stress on healthy cells or conversely, that 2) inhibit telomere repair in malignant ALT tumors to halt proliferation.