Structural and Biological Effects of Ribonucleotide Insertion into Telomeres

项目摘要 端粒是位于染色体末端的保护性非编码DNA帽，维持基因组的完整性。在……里面 大多数体细胞组织的端粒会随着连续几轮的复制而缩短，并将达到临界短 长度，在这一点上，一个细胞将变得衰老或经历凋亡。这种命运是可以避免的，如果端粒 是通过表达端粒酶来维持的，端粒酶是一种逆转录酶，通过增加 端粒重复。端粒的这种延长可以导致复制的永生，这是癌症的标志之一。 癌症的另一个特征是DNA损伤引起的基因组不稳定。最流行的DNA形式 损伤是在DNA复制过程中插入的核糖核苷酸(RNTP)。如果不加以修复，这些rntp将促进 基因组的不稳定性，DNA二级结构的变化，以及人类疾病。考虑到有害的影响 在DNA中的rNTPs中，细胞已经进化出核糖核苷酸切除修复(RER)途径来移除rNTPs。而当 基因组中rNTPs的影响已被公认为具有有害影响并促进人类疾病， RNTPs对端粒的影响尚不清楚。一种重要的DNA二级结构见于端粒， 可受rNTPs影响的是G-四链(G4)。具体到这一提议，目前还不知道会有什么效果 RNTP将对端粒结构和完整性产生影响，或者端粒rNTP如何修复以保护端粒 正直。为了研究这一点，我们开发了一种端粒酶突变体(Y717A)，它能以更大的速度插入rNTPs 比野生型(WT)端粒酶更快。使用这个突变体，我将有选择地提高rNTP的掺入速度 只是在端粒上。这项建议的首要目标是确定rNTPs对端粒G4S的影响 并描述了端粒中rNTPs的修复途径。我假设在端粒中插入rNTP将 改变端粒G4的动力学，并通过RER修复这些RNTP。为了检验这一假设，我建议 具体目的如下：1)表征rNTP插入端粒的结构效应；2) 描述端粒中rNTP修复的特征。对于目标1，我将使用圆二色分光光度来系统地 在体外检测G4的形成。使用将形成G4(TTAGGG)4的基本端粒序列的寡聚，即 将有系统地用rntps取代dntp，以确定rntps是否改变了 端粒。此外，使用含有WT端粒酶或端粒酶突变体的细胞系 RNTPs以高于WT的速度结合G4S特异性抗体来检查是否存在 RNTPs改变端粒的G4含量。对于目标2，我将通过重组RER途径来表征RER 底物具有单一的rNTP，该rNTP要么是线性DNA，要么是折叠成G4的DNA。我会监控 修复产品的形成，并比较不同衬底上的RER功能。此外，使用 邻近依赖的生物素识别(BioID)I将能够识别任何其他涉及的蛋白质 修复端粒上的rNTP。这项研究应用了一种创新的体外生化分析与 细胞分析以检测端粒rNTPs的影响和修复。

Project Abstract Telomeres are protective noncoding DNA caps at the ends of chromosomes that maintain genomic integrity. In most somatic tissues telomeres will shorten with successive rounds of replication and will reach a critically short length, at which point a cell will become senescent or undergo apoptosis. This fate can be avoided if telomeres are maintained through expression of telomerase, a reverse transcriptase that elongates telomeres by adding telomeric repeats. This elongation of telomeres can lead to replicative immortality, one of the hallmarks of cancer. Another hallmark of cancer is genomic instability that arises from DNA damage. The most prevalent form of DNA damage are ribonucleotides (rNTPs) inserted during DNA replication. Left unrepaired these rNTPs will promote genomic instability, changes to the DNA secondary structure, and human diseases. Given the deleterious effects of rNTPs in DNA, cells have evolved the ribonucleotide excision repair (RER) pathway to remove rNTPs. While the impact of rNTPs in the genome is well established to have deleterious effects and promote human disease, the impact of rNTPs at telomeres remains unknown. One essential DNA secondary structure seen at telomeres, that can be affected by rNTPs, is a G-quadruplex (G4). Specific to this proposal, it is not known what effect rNTPs will have on telomeric structure and integrity, or how rNTPs at telomeres are repaired to protect telomere integrity. To investigate this, we have developed a telomerase mutant (Y717A) that inserts rNTPs at a greater rate than wildtype (WT) telomerase. Using this mutant, I will selectively increase the rate of rNTP incorporation only at telomeres. The overarching goal of this proposal is to determine the effect of rNTPs on telomeric G4s and characterize the repair pathway for rNTPs in telomeres. I hypothesize that rNTP insertion into telomeres will alter telomeric G4 dynamics and that these rNTPs are repaired through RER. To test this hypothesis, I propose the following specific aims: 1) characterize the structural effects of rNTP insertion into telomeres and 2) characterize rNTP repair in telomeres. For aim 1, I will use circular dichroism spectrophotometry to systematically examine G4 formation in vitro. Using oligos of the basic telomeric sequence that will form a G4 (TTAGGG)4, I will systematically replace the dNTPs with rNTPs to determine if rNTPs alter the key G4 structural motif in telomeres. Additionally, using cell lines that contain either WT telomerase or a telomerase mutant which inserts rNTPs at a greater rate than WT in conjunction with antibodies that are specific for G4s to examine if the presence of rNTPs alters the G4 content at telomeres. For aim 2, I will characterize RER by reconstituting the RER pathway with a substrate that has a single rNTP that will either be linear DNA or DNA folded into a G4. I will monitor the formation of the repaired product and compare RER functionality on the different substrates. Additionally, using proximity dependent biotin identification (BioID) I will be able to identify any additional proteins that are involved in repair of rNTPs at telomeres. This study applies an innovative combination of in vitro biochemical assays with cellular assays to examine the impact and repair of rNTPs at telomeres.