Topoisomerase 1-catalyzed genomic ribonucleotide excision, its regulation, and its implication in transcription.

拓扑异构酶 1 催化的基因组核糖核苷酸切除、其调节及其在转录中的意义。

• 批准号: 10536476
• 负责人: Edward James Sarrain
• 金额: $ 3.08万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-06 至 2026-09-05
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10536476
• 关键词: Affect; BRCA1 gene; BRCA2 gene; Binding; Biochemical; Biological; Cells; Complex; Coupled; DNA; DNA Adducts; DNA Ligases; DNA Repair; DNA Structure; DNA Topoisomerases; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Data; Deoxyribonucleotides; Enzymes; Eukaryota; Excision; Genes; Genetic Transcription; Genome; Genome Stability; Genomic Instability; Genomics; Human; In Vitro; Investigation; Laboratories; Lead; Lesion; Ligation; Link; Maps; Mediating; Modeling; Molecular; Monitor; Nature; Pathway interactions; Periodicity; Phenotype; Physiological; Poly(ADP-ribose) Polymerases; Polymerase; Proteins; RNA; Regimen; Regulation; Relaxation; Research; Ribonucleases; Ribonucleotides; Role; Single-Stranded DNA; Site; Specificity; Stress; Structure; Superhelical DNA; Testing; Topoisomerase; Torsion; Transcriptional Regulation; Work; Yeasts; adduct; base; cancer therapy; inhibitor; inorganic phosphate; insight; lead phosphate; mRNA Expression; mutant; novel; nuclease; preservation; prevent; reconstitution; recruit; repaired; transcriptome sequencing; tumor

PROJECT SUMMARY In eukaryotes, ribonucleotides are frequently incorporated into DNA during replication (1 ribonucleotide per every 1000-5000 deoxyribonucleotides). Canonically, RNase H2 is the protein responsible for the removal of these embedded ribonucleotides. However, it has been recently shown that topoisomerase 1 (Top1) also has its own genomic ribonucleotide processing activity. When this processing occurs in specific short repeat sequences, it can lead to 2-7 bp deletions. These deletions are the result of two sequential nicks by Top1 that releases a small single-stranded DNA segment and is then followed by a strand slippage and ligation across the formed gap. These deletions have been shown to be biased towards the non-transcribed strand (NTS) of highly transcribed genes, but the reasoning for this strand specificity is yet to be elucidated. With the help of our recent preliminary data, we propose that this strand specificity for Top1 activity is due to the formation of DNA topological structures, specifically negative supercoils, behind the RNA polymerase that bias the initial cleavage by Top1. This would re-define the way we think about Top1-mediated DNA relaxation by limiting the cleavage of Top1 mainly towards the NTS during transcription. This limitation also allowed us to hypothesize about a possible biological implication for Top1-catalyzed excision of ribonucleotides. When Top1 cleaves a ribonucleotide, it can generate a unique nick lesion known as 2’,3’-cyclic phosphate (CP). The bias of Top1 cleavage at the NTS and the formation of CPs lead us to hypothesize that this transient nick lesion could serve as a way of continually relieving transcriptional torsional stress, as CPs would be forming at a strand that would potentially not interfere with transcription, unlike nicks at the transcribing strand. We plan to investigate this by looking at mRNA expression after depleting genomic ribonucleotides in yeast. This investigation will provide us with potential roles of ribonucleotide incorporation in eukaryotes. Additionally, the processing of genomic ribonucleotides by Top1 produces PARP-trapping lesions, a chemotherapeutic target for BRCA1/BRCA2 deficient tumors. I will look at the interactions that could lead to the trapping of PARP1 after ribonucleotide cleavage by Top1 and its link to the ability of PARP1 to regulate CP formation (identified recently by us and shown in our preliminary data). The presence of either CPs or adducts between DNA and Top1 are likely candidates for the recruitment of PARP1. Overall, our findings will help clarify the relevance of ribonucleotide incorporation for transcriptional regulation by providing insight into a novel mechanism of DNA relaxation by Top1 through the processing of those genomic ribonucleotides. Our proposal will also aim to describe the regulation of CP formation by PARP1, and the physiological relevance of the interaction between Top1, PARP1, and genomic ribonucleotides.

项目摘要 在真核生物中，核糖核苷酸在复制过程中经常掺入DNA中（每个细胞中有1个核糖核苷酸）。 1000-5000个脱氧核糖核苷酸）。通常，RNase H2是负责去除这些蛋白质的蛋白质。 嵌入的核糖核苷酸。然而，最近已经表明拓扑异构酶1（Top1）也具有其自身的 基因组核糖核苷酸加工活性。当这种加工发生在特定的短重复序列中时， 可导致2-7 bp缺失。这些缺失是Top1连续两次切割的结果， 单链DNA片段，然后进行链滑移和连接穿过形成的缺口。 这些缺失已经显示偏向于高度转录的非转录链（NTS）。 基因，但这种链特异性的推理尚未阐明。借助我们最近的初步调查 数据，我们提出，这种链特异性Top1活性是由于DNA拓扑结构的形成， 特别是负超螺旋，位于RNA聚合酶后面，使Top1的初始切割偏向。这将 重新定义我们认为Top1介导的DNA松弛的方式，通过限制Top1的切割主要朝向 在转录过程中的NTS。这种局限性也使我们能够假设一种可能的生物学含义 用于Top1催化的核糖核苷酸切除。当Top1切割核糖核苷酸时，它可以产生独特的 称为2 '，3'-环磷酸（CP）的切口损伤。在NTS处Top1切割的偏向性和 CP使我们假设这种短暂的切口损伤可以作为一种持续缓解的方式， 转录扭转应力，因为CP将在可能不干扰转录的链上形成。 转录，不像在转录链切口。我们计划通过观察mRNA表达来研究这一点 在酵母中耗尽基因组核糖核苷酸后。这项调查将为我们提供潜在的作用， 真核生物中的核糖核苷酸掺入。此外，Top1对基因组核糖核苷酸的加工 产生PARP捕获损伤，这是BRCA 1/BRCA 2缺陷肿瘤的化疗靶点。我会看看 可能导致核糖核苷酸被Top1切割后PARP 1被捕获的相互作用及其与 PARP 1调节CP形成的能力（我们最近发现并在我们的初步数据中显示）。的 CP或DNA与Top1之间加合物的存在可能是PARP 1募集的候选物。 总的来说，我们的发现将有助于澄清核糖核苷酸掺入转录调控的相关性， 提供了一个新的机制，DNA松弛的Top1通过处理这些基因组 核糖核苷酸。我们的建议还旨在描述PARP 1对CP形成的调节，以及 Top1，PARP 1和基因组核糖核苷酸之间相互作用的生理相关性。