Processing of Alternative Structures in Telomeric DNA

描述(申请人提供)：端粒对维持染色体的完整性至关重要，端粒功能障碍已成为细胞衰老和可能的干细胞功能丧失的原因，导致组织功能和再生受损。端粒功能障碍与多种与年龄相关的疾病有关，如癌症、动脉粥样硬化和骨质疏松。节段性进展性疾病Werner综合征以端粒随机丢失和细胞早衰为特征，由WRN解旋酶/核酸外切酶蛋白丢失引起。WRN蛋白与端粒蛋白POT1和TRF2相互作用，这两种蛋白都参与端粒末端交替结构的处理，包括G-四链(G4DNA)、t-环/d-环和Holliday连接(HJ)。这种结构是WRN蛋白的首选底物，端粒上这些结构的不当处理可能会导致端粒丢失和基因组不稳定。我们假设在端粒DNA中存在交替的DNA结构处于动态平衡状态，这种平衡是由WRN和端粒结合蛋白共同调节的，我们将使用几种生物物理和生化方法进行测试。在特定的目标1中，我们将测试WRN解旋酶/核酸外切酶和POT1是否在解析端粒G4 DNA方面合作，端粒G4 DNA是DNA聚合酶的障碍。G4DNA的形成和解离将使用单分子荧光共振能量转移(FRET)来跟踪。在具体目标2中，我们将使用原子力显微镜和计算模型来确定G4DNA结构在可能处于多种状态的长端粒DNA分子上的排列和平衡。然后，我们将研究POT1结合是否可以协同破坏较长分子上的G4 DNA的稳定。在特定的目标3中，我们将确定TRF2和POT1在解离HJ结构中调节WRN活性的能力。HJ构建物将由两个端粒和两个独特的序列DNA臂组成，每个臂分别标记一个荧光团。我们独特的荧光标记系统将允许我们在链置换凝胶分析中比较每个端粒手臂与构建的混合序列手臂的命运。公共卫生相关性：端粒DNA结构的不正确处理可能是几种与衰老有关的疾病的基础。这项建议分析了端粒DNA处理的机制，这将为衰老过程提供洞察力，并可能有助于找到阻止或延缓基因组不稳定和端粒丢失的治疗靶点。

DESCRIPTION (provided by applicant): Telomeres are crucial for maintaining the integrity of chromosomes, and telomeric dysfunction has emerged as a cause for cell senescence and possible stem cell loss of function, leading to impaired tissue function and regeneration. Telomere dysfunction has been implicated in diverse age-associated diseases, such as cancer, atherosclerosis and osteoporosis. The segmental progeroid disease Werner Syndrome is characterized by the stochastic loss of telomeres and premature cell senescence, and is caused by loss of the WRN helicase/exonuclease protein. WRN protein interacts with telomeric proteins POT1and TRF2, which are both implicated in the processing of alternate structures at telomeric ends, including G-quadruplexes (G4 DNA), t-loop/d-loops, and Holliday Junctions (HJ). Such structures are preferred substrates for WRN protein, and improper processing of these structures at telomeres can lead to telomere loss and genomic instability. We hypothesize that alternate structures of DNA exist in dynamic equilibrium in telomeric DNA and the equilibrium is modulated by WRN in cooperation with telomere-binding proteins, which we will test using several biophysical and biochemical methods. In specific aim 1, we will test whether WRN helicase/exonuclease and POT1 cooperate in resolving telomeric G4 DNA, an impediment to DNA polymerase. G4 DNA formation and dissociation will be tracked using single molecule fluorescence resonance energy transfer (FRET). In specific aim 2, we will use Atomic Force Microscopy and computational modeling to determine the arrangement and equilibrium of G4 DNA structures on long telomeric DNA molecules in which multiple states are possible. We will then study whether POT1 binding can cooperatively destabilize G4 DNA on longer molecules. In specific aim 3, we will determine the ability of TRF2 and POT1 to modulate WRN activity in dissociating HJ structures. The HJ constructs will be composed of two telomeric and two unique-sequence DNA arms, with each arm individually labeled with a fluorophore. Our unique fluorophore-labeling system will permit us to compare the fates of each telomeric arm relative to the constructs mixed-sequence arms in strand displacement gel assays. PUBLIC HEALTH RELEVANCE: Improper processing of telomeric DNA structures may underlie several aging-related diseases. This proposal analyzes the mechanisms for telomeric DNA processing, which should provide insights into the aging process and may assist in finding therapeutic targets to arrest or delay genomic instability and telomere loss.