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，大约13%的 人类基因组包含可形成非典型或非B DNA构象序列基序。这 该项目的重点是G-四链体，这是一种非B DNA，我们有最有力的证据表明， 人类细胞中的全基因组形成和功能。有超过70万的假定 人类基因组中的G-四链体基因座。它们占基因组的~1%，相比之下， 蛋白质编码外显子。最近的体内实验表明，G-四链体调节关键的细胞过程 (e.g.,染色质组织和转录）。因此，我们假设某些G-四链体基因座 在净化选择下进化。然而，G-四链体可能是DNA复制的障碍。我们 基于对长读序数据的分析，已发表的初步结果表明， 聚合速度和增加的聚合错误在G-四链体基因座全基因组。我们 假设同样的现象发生在人类细胞中，并导致突变增加， G-四链体基因座在我们已发表和未发表的初步结果的基础上，该项目将 研究G-四链体基序对基因组进化的贡献，这一直是至关重要的 探索不足目的1将阐明G-四链体突变率增加背后的机制基础 基因座，使用最先进的高保真双链测序。通过体内实验，我们将验证一个假设 突变率特别是在人类细胞中形成的G-四链体结构中增加， 与复制减慢相关。通过体外实验，我们将检验一个假设， 真核生物复制聚合酶（聚合酶β和δ，负责前导和滞后链 合成）失速并且在G-四路复用器处具有增加的错误频率。目标2将评估 G-四链体基因座对基因组突变率区域变异的贡献，并将测试一个 G-四链体基因座促进人类群体和染色体结构变异假说 进化过程中的重组高级统计技术，包括来自Functional Data的技术 分析域，将用于此目的。最后，目标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测试。我们将测试一个假设，即G-四链体位于不同的 基因组的功能区室经历不同的选择压力，例如，启动子基序是 在强烈的净化选择下进化。此外，我们将调查一个潜在的关联 G-四链体结构的生物物理稳定性和作用于它们的选择强度之间的关系。这 目的还将确定生理相关的G-四链体基因座，这将驱动未来的功能 问题研究总的来说，该项目将大大促进我们对 G-四链体与基因组进化和疾病。