The impact of G-quadruplexes on genome evolution

项目总结 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规范或非B类DNA构象的序列基序。这 该项目的重点是G-四链，这是我们拥有最有力证据的非B-DNA类型 人类细胞的全基因组结构和功能。有超过70万名推定的 人类基因组中的G-四链基因座。它们占基因组的~1%，相比之下，占基因组的~1.5% 蛋白质编码外显子。最近的活体实验表明，G-四联体调节关键的细胞过程 (例如，染色质组织和转录)。因此，我们假设一些G-四联体基因座 在净化选择下进化。然而，G-四联体可能是DNA复制的障碍。我们的 发表的初步结果，基于对长时间测序数据的分析，显示出下降 全基因组G-四链基因座聚合速度和聚合误差增加。我们 假设同样的现象发生在人类细胞中，并导致在 G-四联体基因座。在我们已公布和未公布的初步结果的基础上，该项目将 研究G-四链基序对基因组进化的贡献，这一点一直是至关重要的 开发不足。目标1将阐明G-四链突变率增加的机制基础 基因座，使用最先进的高保真双链测序。通过活体实验，我们将检验一个假设 这种突变率特别是在人类细胞中形成的G-四链结构中增加，并且是 与复制速度减慢相关。通过体外实验，我们将检验一个假设，即两个主要的 真核复制聚合酶(聚合酶epsilon和Delta，负责领先和滞后链 分别合成)停滞，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-四联体基因座对基因组突变率的区域变异的贡献，并将测试 G-四链基因座促进人类群体和染色体结构变异的假说 进化过程中的重排。高级统计技术，包括来自功能数据的技术 分析领域，将用于这一目标。最后，目标3将研究作用于G-四链基因座的选择 使用经典的和新颖的统计检验。我们将检验一个假设，即G-四链体位于不同的 基因组的功能间隔经历了不同的选择压力，例如，启动子基序是 预计将在强净化选择下进化。此外，我们将调查一个潜在的关联 G-四链结构的生物物理稳定性与作用于其上的选择强度之间的关系。这 AIM还将识别一组生理上相关的G-四链基因座，这些基因座将驱动未来的功能性 学习。总体而言，该项目将极大地促进我们对 G-四联体与基因组进化和疾病的关系。

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 Building upon our published and unpublished preliminary results, this project will examine the contribution of G-quadruplex motifs to genome evolution, which has been critically underexplored.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