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

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 是负责去除这些物质的蛋白质。 嵌入核糖核苷酸。然而，最近的研究表明，拓扑异构酶 1 (Top1) 也有自己的 基因组核糖核苷酸加工活性。当这种处理发生在特定的短重复序列中时， 可能导致 2-7 bp 缺失。这些删除是 Top1 两个连续切口的结果，释放了一个小的 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，但这种链特异性的原因尚未阐明。在我们最近的初步研究的帮助下 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。这会 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 转录过程中的NTS。这种限制也使我们能够假设可能的生物学意义 用于 Top1 催化的核糖核苷酸切除。当Top1切割核糖核苷酸时，它可以产生独特的 称为 2',3'-环磷酸盐 (CP) 的切口损伤。 NTS 处 Top1 裂解的偏差和形成 CP 使我们推测这种短暂的切口损伤可以作为持续缓解疼痛的一种方法。 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 耗尽酵母中的基因组核糖核苷酸后。这项调查将为我们提供以下潜在的角色： 真核生物中核糖核苷酸的掺入。此外，Top1 对基因组核糖核苷酸的加工 产生 PARP 捕获病灶，这是 BRCA1/BRCA2 缺陷肿瘤的化疗靶点。我会看看 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（我们最近确定并在我们的初步数据中显示）。的 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 核糖核苷酸。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。

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.