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。

基本信息

批准号：
10704085
负责人：
Edward James Sarrain
金额：
$ 3.17万
依托单位：
TRUSTEES OF INDIANA UNIVERSITY
依托单位国家：
美国
项目类别：
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/10704085
关键词：
Affect BRCA1 gene BRCA2 gene Binding Biochemical Biological Cells Chromosome Mapping Complex Coupled DNA DNA Adduction DNA Adducts DNA Ligases DNA Repair DNA Structure DNA-Directed DNA Polymerase DNA-Directed RNA Polymerase Data Deoxyribonucleotides Enzymes Eukaryota Excision Genetic Transcription Genome Genome Stability Genomic Instability Genomics Human In Vitro Investigation Laboratories Lesion Ligation Link Mediating Modeling Molecular Monitor Nature Pathway interactions Periodicity Phenotype Physiological Poly(ADP-ribose) Polymerase Inhibitor Poly(ADP-ribose) Polymerases Polymerase Proteins RNA Regimen Regulation Relaxation Research Ribonucleases Ribonucleotides Role Single-Stranded DNA Site Specificity Stress Structure Superhelical DNA Testing Therapeutic Topoisomerase Transcriptional Regulation Work Yeasts adduct cancer therapy inorganic phosphate insight lead phosphate mRNA Expression mutant novel nuclease preservation prevent reconstitution recruit repaired therapeutic target transcriptome sequencing tumor

项目摘要

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.

项目概要在真核生物中，核糖核苷酸在复制过程中经常掺入 DNA 中（每个核糖核苷酸 1 个） 1000-5000 个脱氧核糖核苷酸）通常，RNase H2 是负责去除这些的蛋白质。然而，最近发现拓扑异构酶 1 (Top1) 也有自己的结构。当这种加工发生在特定的短重复序列中时，它会影响基因组核糖核苷酸加工活性。可导致 2-7 bp 缺失，这些缺失是 Top1 释放一个小片段的两个连续切口的结果。单链 DNA 片段，然后进行链滑移并跨过形成的间隙连接。这些缺失已被证明偏向于高度转录的非转录链（NTS）基因，但在我们最近的初步研究的帮助下，这种链特异性的原因尚未阐明。根据数据，我们认为 Top1 活性的这种链特异性是由于 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 的初始切割产生偏差。通过限制 Top1 的裂解，重新定义我们对 Top1 介导的 DNA 松弛的思考方式这种限制也让我们能够争论可能的生物学意义。用于 Top1 催化的核糖核苷酸切除当 Top1 切割核糖核苷酸时，它可以生成独特的核苷酸。被称为 2',3'-环磷酸盐 (CP) 的切口损伤 NTS 处 Top1 裂解的偏差以及 2',3'-环磷酸盐的形成。 CP 带领我们战斗，这种短暂的切口损伤可以作为持续缓解疼痛的一种方式转录扭转应力，因为 CP 将在一条可能不会干扰的链上形成转录，与转录链上的切口不同，我们计划通过观察 mRNA 表达来研究这一点。在耗尽酵母中的基因组核糖核苷酸后，这项研究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 捕获病变，这是 BRCA1/BRCA2 缺陷肿瘤的化疗靶标。核糖核苷酸被 Top1 切割后可能导致 PARP1 捕获的相互作用及其与 PARP1 调节 CP 形成的能力（我们最近确定并显示在我们的初步数据中）。 DNA 和 Top1 之间存在 CP 或加合物可能是 PARP1 招募的候选者。总的来说，我们的研究结果将有助于阐明核糖核苷酸掺入与转录调控的相关性通过对这些基因组的处理，深入了解 Top1 的 DNA 松弛新机制我们的提案还旨在描述 PARP1 对 CP 形成的调节，以及 Top1、PARP1 和基因组核糖核苷酸之间相互作用的生理相关性。