Nature Communications：RPA将RNase H1转化为双向外切核糖核酸酶，用于RNA-DNA杂交切割-动脉网

Nature Communications：RPA transforms RNase H1 to a bidirectional exoribonuclease for processive RNA–DNA hybrid cleavage

Nature 等信源发布 2024-08-29 10:15

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AbstractRNase H1 has been acknowledged as an endoribonuclease specializing in the internal degradation of the RNA moiety within RNA–DNA hybrids, and its ribonuclease activity is indispensable in multifaceted aspects of nucleic acid metabolism. However, the molecular mechanism underlying RNase H1-mediated hybrid cleavage remains inadequately elucidated.

摘要RNA酶H1被认为是一种内切核糖核酸酶，专门用于RNA-DNA杂种中RNA部分的内部降解，其核糖核酸酶活性在核酸代谢的多方面是必不可少的。然而，RNase H1介导的杂交切割的分子机制仍未得到充分阐明。

Herein, using single-molecule approaches, we probe the dynamics of the hybrid cleavage by Saccharomyces cerevisiae RNase H1. Remarkably, a single RNase H1 enzyme displays 3′-to-5′ exoribonuclease activity. The directional RNA degradation proceeds processively and yet discretely, wherein unwinding approximately 6-bp hybrids as a prerequisite for two consecutive 3-nt RNA excisions limits the overall rate within each catalytic cycle.

在此，使用单分子方法，我们探测了酿酒酵母RNase H1杂交切割的动力学。值得注意的是，单个RNase H1酶显示3'-5'核糖核酸外切酶活性。定向RNA降解进行过程性但不连续，其中解开大约6 bp的杂种作为两个连续的3-nt RNA切除的先决条件限制了每个催化循环内的总体速率。

Moreover, Replication Protein A (RPA) reinforces RNase H1’s 3′-to-5′ nucleolytic rate and processivity and stimulates its 5′-to-3′ exoribonuclease activity. This stimulation is primarily realized through the pre-separation of the hybrids and consequently transfers RNase H1 to a bidirectional exoribonuclease, further potentiating its cleavage efficiency.

此外，复制蛋白A（RPA）增强了RNase H1的3'-to-5'核酸溶解速率和持续合成能力，并刺激了其5'-to-3'核酸外切酶活性。这种刺激主要通过杂交体的预分离来实现，因此将RNase H1转移到双向核糖核酸外切酶上，进一步提高了其切割效率。

These findings unveil unprecedented characteristics of an RNase and provide a dynamic view of RPA-enhanced processive hybrid cleavage by RNase H1..

这些发现揭示了RNase前所未有的特征，并提供了RNase H1对RPA增强的进行性杂交切割的动态视图。。

IntroductionNucleases are a family of phosphodiesterases tasked with catalyzing the cleavage of the phosphodiester bonds that link nucleotides within nucleic acids1. Based on the cleavage mode, nucleases can be classified into endonucleases and exonucleases. The main distinction lies in that endonucleases internally introduce cleavage sites along nucleic acids, whereas exonucleases continuously digest nucleic acids from their ends, producing mononucleotides or oligonucleotides as products1,2.

引言核酸酶是磷酸二酯酶家族，其任务是催化连接核酸内核苷酸的磷酸二酯键的裂解1。根据切割模式，核酸酶可分为核酸内切酶和核酸外切酶。。

RNase H are a class of evolutionarily conserved nucleases that appear ubiquitous in bacteria, archaea, and eukaryotes3,4. Since their discovery, RNase H have been widely recognized as endoribonucleases that specifically catalyze the elimination of the RNA moiety of RNA–DNA hybrids and R-loops in a sequence non-specific manner5,6.

RNase H是一类进化上保守的核酸酶，在细菌，古细菌和真核生物中普遍存在3,4。自从他们发现以来，RNase H被广泛认为是内切核糖核酸酶，它以序列非特异性方式特异性催化消除RNA-DNA杂种和R环的RNA部分5,6。

RNase H are generally involved in the maintenance of genome stability, such as R-loop suppression, DNA replication, homologous recombination (HR)-directed DNA double-strand break (DSB) repair, and ribonucleotide excision repair. Their significance is underscored by the genetic linkage to defective mitochondrial DNA replication and a severe neurological disorder called Aicardi-Goutières syndrome (AGS)7,8.

RNase H通常参与维持基因组稳定性，例如R环抑制，DNA复制，同源重组（HR）定向的DNA双链断裂（DSB）修复和核糖核苷酸切除修复。线粒体DNA复制缺陷和一种称为Aicardi-Goutières综合征（AGS）7,8的严重神经系统疾病的遗传联系突显了它们的重要性。

Among their multifaceted cellular functions, RNase H must deal with hybrids and R-loops with distinct lengths, from single base to several kilobase pairs9,10,11. Many vital functions and properties of RNase H have been brought to light in the past few decades3,4,7,12,13. However, one intriguing question that remains unanswered is how RNase H at a physiological concentration of a few nanomolar range effectively degrade a long stretch of an RNA–DNA hybrid or an R-loop in cells14.Based on the sequence similarities and biochemical properties, eukaryotic RNase .

在其多方面的细胞功能中，RNase H必须处理具有不同长度的杂种和R环，从单碱基到几千碱基对9,10,11。在过去的几十年中，RNase H的许多重要功能和特性已经被揭示出来3,4,7,12,13。然而，一个尚未解决的有趣问题是，生理浓度为几纳摩尔范围的RNase H如何有效降解细胞中长链的RNA-DNA杂种或R环14。基于序列相似性和生化特性，真核RNase。

Data availability

数据可用性

All data supporting the findings of this study are available within the paper and its Supplementary Information. Source data are provided with this paper.

本文及其补充信息中提供了支持本研究结果的所有数据。本文提供了源数据。

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Download referencesAcknowledgementsWe thank all the staff of the molecular and cell biology core facility of the School of Life Science and Technology at ShanghaiTech University for providing technical support. This work was supported by the National Natural Science Foundation of China (32022048 and 32271505 to B.S., 32230029 and 81925015 to W.L.), the Natural Science Foundation of Shanghai (22ZR1441900 and 23ZR1442200 to B.S.), and ShanghaiTech University School of Life Science and Technology Development Fund (to B.S.).Author informationAuthor notesThese authors contributed equally: Yanan Li, Chao Liu.Authors and AffiliationsSchool of Life Science and Technology, ShanghaiTech University, Shanghai, ChinaYanan Li, Xinshuo Jia, Lulu Bi, Zhiyun Ren, Yilin Zhao, Xia Zhang, Lijuan Guo, Yanling Bao & Bo SunGuangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou, ChinaChao Liu & Wei LiState Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, ChinaChao Liu & Wei LiUniversity of Chinese Academy of Sciences, Beijing, ChinaZhiyun Ren & Wei LiInterdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, ChinaCong LiuAuthorsYanan LiView author publicationsYou can also search for this author in.

下载参考文献致谢我们感谢上海理工大学生命科学与技术学院分子与细胞生物学核心设施的所有工作人员提供的技术支持。这项工作得到了国家自然科学基金（32022048和32271505授予B.S.，32230029和81925015授予W.L.），上海自然科学基金（22ZR1441900和23ZR1442200授予B.S.）和上海理工大学生命科学与技术发展基金（授予B.S.）的支持。作者信息作者注意到这些作者做出了同样的贡献：李亚南，刘超。作者和附属机构上海理工大学生命科学与技术学院，上海，中国李亚楠，贾心硕，毕露露，任志云，赵依林，张霞，郭丽娟，鲍艳玲，鲍双光，广州医科大学妇女儿童医学中心，广州，中国刘朝伟中国科学院动物研究所干细胞与生殖生物学国家重点实验室，北京，中国刘朝伟中国科学院大学，北京，中国任志云，李朝伟中国科学院上海有机化学研究所生物与化学跨学科研究中心，上海，中国刘聪Orsyanan LiView作者出版物您也可以在中搜索这位作者。

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PubMed Google ScholarContributionsB.S. and W.L. conceived the project and supervised all research. Y.L. and Ch.L. conducted all the experiments. Y.L. Ch.L., W.L., and B.S. analyzed and interpreted the data. X.J., Z.R., Y.Z., X.Z., L.B., Y.B., L.G., and Co.L. helped with the protein purification and hybrid template preparation.

PubMed谷歌学术贡献b。S、。Y、 L.和Ch.L.进行了所有实验。Y、 L.Ch.L.，W.L。和B.S.分析并解释了数据。十、 J.，Z.R.，Y.Z.，X.Z.，L.B.，Y.B.，L.G。和Co.L.帮助蛋白质纯化和杂交模板制备。

B.S. Y.L., Ch.L., and W.L. wrote the manuscript with inputs from all authors.Corresponding authorsCorrespondence to.

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Nature Communications thanks Shixin Liu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

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Reprints and permissionsAbout this articleCite this articleLi, Y., Liu, C., Jia, X. et al. RPA transforms RNase H1 to a bidirectional exoribonuclease for processive RNA–DNA hybrid cleavage.

转载和许可本文引用本文Li，Y.，Liu，C.，Jia，X。等人。RPA将RNase H1转化为双向核糖核酸酶，用于进行RNA-DNA杂交切割。

Nat Commun 15, 7464 (2024). https://doi.org/10.1038/s41467-024-51984-5Download citationReceived: 04 January 2024Accepted: 21 August 2024Published: 29 August 2024DOI: https://doi.org/10.1038/s41467-024-51984-5Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard.

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