The impact of G-quadruplexes on genome evolution

G-四链体对基因组进化的影响

• 批准号: 10319020
• 负责人: KATERYNA MAKOVA
• 金额: $ 56.79万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10319020
• 关键词: Amyotrophic Lateral Sclerosis; B-DNA; Biophysics; Cell physiology; Cells; Chromatin; Chromosomal Rearrangement; Code; DNA; DNA Sequence; DNA Structure; DNA analysis; DNA biosynthesis; DNA replication fork; Data; Data Analyses; Data Set; Development; Disease; Etiology; Evolution; Exons; Frequencies; Future; G-Quartets; Genetic Models; Genetic Polymorphism; Genetic Transcription; Genome; Genomic Instability; Genomics; Germ-Line Mutation; Goals; Guanine; Human; Human Genome; In Vitro; Investigation; Kinetics; Lead; Light; Link; Malignant Neoplasms; Modeling; Molecular; Molecular Conformation; Mutagenesis; Mutation; Physiological; Play; Point Mutation; Polymerase; Population; Population Genetics; Process; Proteins; Publishing; Reporting; Role; Running; Site; Source; Speed; Statistical Data Interpretation; Structure; Subjects Selections; Techniques; Technology; Testing; Variant; base; cancer type; experience; experimental study; genome-wide; improved; in vivo; inter-individual variation; mammalian genome; molecular phenotype; novel; polymerization; pressure; promoter

PROJECT SUMMARY Apart from the double-helix B-DNA structure discovered by Watson and Crick, approximately 13% of the human genome comprises sequence motifs that can form non-canonical, or non-B, DNA conformations. This project focuses on G-quadruplexes, the type of non-B DNA for which we have the strongest evidence of genome-wide formation and functionality in human cells. There are more than 700,000 putative G-quadruplex loci in the human genome. They constitute ~1% of the genome, compared to ~1.5% occupied by protein-coding exons. Recent in vivo experiments showed that G-quadruplexes regulate key cellular processes (e.g., chromatin organization and transcription). Thus we hypothesize that some groups of G-quadruplex loci evolve under purifying selection. Yet, G-quadruplexes may represent a hurdle for DNA replication. Our published preliminary results, based on the analysis of long-read sequencing data, demonstrated decreased polymerization speed and increased polymerization errors at G-quadruplex loci genome-wide. We hypothesize that the same phenomena occur in human cells and lead to increased mutagenesis at G-quadruplex loci. Building upon our published and unpublished preliminary results, this project will examine the contribution of G-quadruplex motifs to genome evolution, which has been critically underexplored. Aim 1 will elucidate the mechanistic basis behind the increased mutation rate at G-quadruplex loci, using state-of-the-art high-fidelity duplex sequencing. With in vivo experiments, we will test a hypothesis that mutation rates are increased specifically at G-quadruplex structures forming in human cells and are associated with replication slowdown. With in vitro experiments, we will test a hypothesis that two major eukaryotic replicative polymerases (polymerases epsilon and delta, responsible for leading and lagging strand synthesis, respectively) stall and have increased error frequencies at G-quadruplexes. Aim 2 will assess the contribution of G-quadruplex loci to regional variation in mutation rates in the genome and will test a hypothesis that G-quadruplex loci facilitate structural variation in human populations and chromosomal rearrangements during evolution. Advanced statistical techniques, including ones from the Functional Data Analysis domain, will be used in this Aim. Finally, Aim 3 will examine selection acting on G-quadruplex loci using classical and novel statistical tests. We will test a hypothesis that G-quadruplexes located in different functional compartments of the genome experience varying selective pressures, e.g., promoter motifs are expected to evolve under strong purifying selection. Moreover, we will investigate a potential association between biophysical stability of G-quadruplex structures and the strength of selection acting on them. This Aim will also identify groups of physiologically relevant G-quadruplex loci that will drive future functional studies. Overall, the project will substantially advance our understanding of the contribution of G-quadruplexes to genome evolution and diseases.

项目摘要 除了沃森和克里克发现的双螺旋B-DNA结构外，约有13％ 人基因组包括可以形成非典型或非B的DNA构象的序列基序。这 项目侧重于G-四链体，这是我们最有力证据的非B DNA类型 人类细胞中全基因组的形成和功能。有超过700,000个推定 人类基因组中的G-四链体基因座。它们占基因组的约1％，而约1.5％ 蛋白质编码外显子。最近的体内实验表明，G四链体调节关键细胞过程 （例如，染色质组织和转录）。因此，我们假设某些G-四链体基因座 在纯化选择下进化。然而，g-四链体可能代表了DNA复制的障碍。我们的 基于长阅读测序数据的分析，已发布的初步结果证明了下降 G-四链体基因座基因组的聚合速度和增加的聚合误差。我们 假设同一现象发生在人类细胞中，并导致诱变增加 G Quadruplex基因座。在我们发表的未发表的初步结果的基础上，该项目将 检查G四链体基序对基因组进化的贡献，这是至关重要的 尚未得到充实的。 AIM 1将阐明G四链体时突变率提高的机理基础 基因座，使用最先进的高保真双工测序。通过体内实验，我们将检验一个假设 在人类细胞中形成的G-四链体结构上，突变速率是特异性提高的，并且是 与复制放缓相关。通过体外实验，我们将测试两个主要的假设 真核复制性聚合酶（聚合酶Epsilon和Delta，负责领先和滞后链 分别综合）失速，在G四链体处的误差频率增加。 AIM 2将评估 G-四链体基因座对基因组突变率的区域变化的贡献，并将测试A g四链体基因座有助于人类和染色体的结构变化的假设 进化过程中的重排。高级统计技术，包括功能数据的技术 分析域将用于此目标。最后，AIM 3将检查作用于G-​​四链体基因座的选择 使用经典和新颖的统计检验。我们将检验一个假设，即G-四链体位于不同 基因组经验的功能隔室各种选择性压力，例如，启动子图案是 预计将在强烈的纯化选择下进化。此外，我们将调查潜在的关联 在g四链体结构的生物物理稳定性与作用于它们的选择强度之间。这 AIM还将识别生理相关的G Qu-Quadlexleclouplex基因座，这些基因座将推动未来的功能 研究。总体而言，该项目将大大提高我们对 G四链体向基因组进化和疾病。