Dissecting the impact of RNA on DNA replication and chromatin structure

剖析 RNA 对 DNA 复制和染色质结构的影响

• 批准号: 10356027
• 负责人: Duncan J Smith
• 金额: $ 60.01万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10356027
• 关键词: Address; Affect; Biochemistry; Biological Assay; Biological Process; Cell division; Cells; Chromatin; Chromatin Structure; Conflict (Psychology); DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Structure; DNA biosynthesis; DNA replication fork; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Discrimination; Enzymes; Etiology; Eukaryota; Eukaryotic Cell; Excision; Excision Repair; Genes; Genetic; Genetic Transcription; Genome; Genomic Instability; Higher Order Chromatin Structure; Human; Impairment; In Vitro; Lesion; Link; Maps; Methods; Modeling; Molecular; Movement; Nuclear RNA; Nucleosomes; Pathway interactions; RNA; Rare Diseases; Resolution; Ribonucleases; Ribonucleosides; Ribonucleotides; Saccharomyces cerevisiae; Saccharomycetales; Site; Source; Structure; Work; genome integrity; genome-wide; human disease; insight; novel; pseudotoxoplasmosis syndrome; repaired; tripolyphosphate; tumor progression

PROJECT SUMMARY Collisions between the DNA replication and transcription machineries (replication-transcription conflicts) appear to be common in eukaryotic cells. Although these conflicts have long been studied as a potential source of DNA damage and, therefore, a threat to genome integrity, we lack a detailed molecular understanding of how the presence of transcribing RNA polymerases on DNA affects the progress of replication, and of the mechanism(s) by which these replication-transcription conflicts give rise to DNA damage. Another consequence of transcription during DNA replication is elevated levels of ribonucleoside triphosphates (rNTPS) – the substrate for RNA polymerases. Due to incomplete discrimination between rNTPs and dNTPs by the replicative DNA polymerases, large numbers of ribonucleotides are mis-incorporated into the genome during each round of replication; this burden is estimated at > 1 million ribonucleotides per cell division in human cells, making ribonucleotides by far the most abundant lesion in eukaryotic DNA. Mis-incorporated ribonucleotides are removed via the ribonucleotide excision repair (RER) pathway, and impaired removal is linked to several human diseases. However, it is not known how ribonucleotides impact chromatin – the higher-order structure of DNA – or conversely how chromatin affects RER. Furthermore, it has not been determined whether all ribonucleotides are equally amenable to repair or what may underlie differences in RER efficiency through the genome. The proposed work encompasses two ongoing projects: The first project addresses how orientation-dependent effects on replication progression and genome integrity arise at transcribed genes. To achieve this, we use a recently developed quantitative method to assay the movement of the replisome genome-wide at high resolution, in combination with genome-wide interrogation of DNA double-strand break formation and a novel assay to map nascent DNA strands in the context of an arrested replication fork. The second project uses a combination of genome-wide assays and in vitro biochemistry to delineate how ribonucleotides destabilize nucleosomes (the basic repeating unit of chromatin), how nucleosomes affect RER initiation by the RNase H2 enzyme, and to elucidate the dynamics of RER at all loci in the genome. The machineries responsible for DNA replication, transcription, and DNA repair are highly conserved throughout eukaryotes. Both projects will be carried out in the budding yeast Saccharomyces cerevisiae: the small genome, rapid replication, and genetic manipulability of S. cerevisiae make this an ideal model in which to study the intersection of fundamental biological processes. Therefore, the results of this work will provide molecular insights into genome instability in humans, and will be directly applicable to our understanding of the etiology and progression of cancer as well as rare diseases including Aicardi-Goutières syndrome.

项目概要 DNA复制和转录机器之间出现冲突（复制-转录冲突） 在真核细胞中很常见。尽管这些冲突长期以来一直被作为 DNA 的潜在来源进行研究 损害，因此对基因组完整性构成威胁，我们缺乏对如何破坏基因组的详细分子了解 DNA 上转录 RNA 聚合酶的存在会影响复制的进程及其机制 这些复制-转录冲突会导致 DNA 损伤。 DNA 复制过程中转录的另一个结果是核糖核苷三磷酸水平升高 (rNTPS) – RNA 聚合酶的底物。由于 rNTP 和 dNTP 之间的不完全区分 在复制DNA聚合酶的作用下，大量核糖核苷酸在复制过程中被错误地掺入基因组中。 每轮复制；据估计，人类细胞中每次细胞分裂产生> 100万个核糖核苷酸， 使核糖核苷酸成为迄今为止真核DNA中最丰富的损伤。错误掺入的核糖核苷酸是 通过核糖核苷酸切除修复 (RER) 途径去除，去除受损与多种人类相关 疾病。然而，目前尚不清楚核糖核苷酸如何影响染色质（DNA 的高阶结构） 或者相反染色质如何影响 RER。此外，尚未确定是否所有核糖核苷酸 同样易于修复或可能导致基因组 RER 效率差异的原因。 拟议的工作包括两个正在进行的项目： 第一个项目解决方向依赖性如何影响复制进程和基因组完整性 出现在转录基因处。为了实现这一目标，我们使用最近开发的定量方法来测定 复制体在全基因组范围内以高分辨率移动，并结合全基因组询问 DNA 双链断裂形成和一种在被捕背景下绘制新生 DNA 链的新方法 复制叉。 第二个项目结合了全基因组测定和体外生物化学来描述如何 核糖核苷酸破坏核小体（染色质的基本重复单位）的稳定性，核小体如何影响 RER 由 RNase H2 酶启动，并阐明基因组中所有位点的 RER 动态。 负责 DNA 复制、转录和 DNA 修复的机器在整个过程中都高度保守。 真核生物。这两个项目都将在芽殖酵母酿酒酵母中进行：小基因组， 酿酒酵母的快速复制和遗传可操作性使其成为研究酿酒酵母的理想模型 基本生物过程的交叉点。因此，这项工作的结果将为分子 对人类基因组不稳定性的见解，将直接应用于我们对病因学的理解 癌症的进展以及罕见疾病，包括 Aicardi-Goutières 综合征。