The impact of G-quadruplexes on genome evolution

G-四链体对基因组进化的影响

基本信息

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
项目类别：
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

项目摘要

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 Building upon our published and unpublished preliminary results, this project will examine the contribution of G-quadruplex motifs to genome evolution, which has been critically underexplored.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.

项目概要除了沃森和克里克发现的双螺旋 B-DNA 结构外，大约 13% 人类基因组包含可以形成非规范或非 B 型 DNA 构象的序列基序。这项目重点关注 G-四链体，这是一种非 B DNA 类型，我们对此拥有最有力的证据人类细胞中全基因组的形成和功能。推定数量超过 700,000 人类基因组中的 G-四联体基因座。它们约占基因组的 1%，而约占基因组的 1.5% 蛋白质编码外显子。最近的体内实验表明 G-四链体调节关键的细胞过程（例如，染色质组织和转录）。因此，我们假设某些 G-四链体基因座组在净化选择下进化。然而，G-四链体可能是 DNA 复制的障碍。我们的根据对长读长测序数据的分析，发表的初步结果表明，全基因组 G 四链体基因座的聚合速度和聚合错误增加。我们假设相同的现象发生在人类细胞中并导致突变增加 G-四联体基因座。基于我们已发表和未发表的初步结果，该项目将检查 G-四链体基序对基因组进化的贡献，这已被批判性地尚未充分探索。目标 1 将阐明 G-四链体突变率增加背后的机制基础位点，使用最先进的高保真双链测序。通过体内实验，我们将检验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，并且是与复制速度减慢有关。通过体外实验，我们将检验两个主要的假设：真核复制聚合酶（聚合酶 epsilon 和 delta，负责前导链和滞后链合成）在 G 四链体上停滞并增加了错误频率。目标 2 将评估 G-四联体基因座对基因组突变率区域变异的贡献，并将测试 G-四联体基因座促进人类群体和染色体结构变异的假设进化过程中的重新排列。先进的统计技术，包括来自功能数据的技术分析域，将用于此目的。最后，目标 3 将检查作用于 G-四联体基因座的选择使用经典和新颖的统计测试。我们将检验 G-四联体位于不同位置的假设基因组的功能区室经历不同的选择压力，例如，启动子基序是预计在强纯化选择下进化。此外，我们将调查潜在的关联 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的关系。这 Aim 还将确定生理相关的 G-四链体基因座组，这些基因座将驱动未来的功能研究。总体而言，该项目将极大地增进我们对以下贡献的理解： G-四链体与基因组进化和疾病。