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: 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
• 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; 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

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（每个核糖核苷酸1 1000-5000脱氧核糖核苷酸）。在规范上，RNase H2是负责去除这些蛋白的蛋白质 嵌入的核糖核苷酸。但是，最近已经证明拓扑异构酶1（top1）也有自己的 基因组核糖核苷酸加工活性。当此处理以特定的短重复序列发生时， 可以导致2-7 bp删除。这些删除是两个顺序划线的结果，top1释放了一个小的 单链DNA段，然后在形成的间隙上进行链条滑移和连接。 这些删除已被证明偏向于高度转录的非转录链（NTS） 基因，但是这种链特异性的推理尚待阐明。在我们最近的初步的帮助下 数据，我们建议TOP1活性的这种链特异性是由于DNA拓扑结构的形成， 特别是负性超元，在RNA聚合酶后面，将初始裂解偏向TOP1。这会 通过将TOP1的裂解主要限制为top1介导的DNA放松，重新定义了我们对Top1介导的DNA放松 转录过程中的NTS。这种限制也使我们可以假设可能的生物学含义 用于核糖核苷酸的Top1催化惊喜。当Top1裂解核糖核苷酸时，它可以生成唯一的 尼克病变称为2'，3'周期磷酸盐（CP）。 top1裂解在NTS的偏置和形成 CPS导致我们假设这种短暂的尼克病变可以作为一种不断缓解的方式 转录扭转应力，因为CP会在链中形成，这可能不会干扰 转录，与抄录链的划痕不同。我们计划通过查看mRNA表达来调查这一点 在耗尽酵母中的基因组色带后。这项投资将为我们提供潜在的作用 真核生物中的核糖核苷酸掺入。另外，TOP1处理基因组核糖核苷酸 产生PARP捕获病变，这是BRCA1/BRCA2缺乏肿瘤的化学治疗靶标。我会看 核糖核苷酸裂解后，可能导致PARP1捕获的相互作用，top1及其链接与 PARP1调节CP形成的能力（美国最近确定并在我们的初步数据中显示）。 DNA和TOP1之间的CPS或加合物的存在可能是募集PARP1的候选者。 总体而言，我们的发现将有助于确定通过 通过处理那些基因组，通过TOP1提供了对DNA松弛的新型机制的见解 核糖核苷酸。我们的建议还将旨在描述PARP1对CP形成的调节，以及 TOP1，PARP1和基因组核苷酸之间相互作用的生理相关性。