Ribose-seq profile and analysis of ribonucleotides in DNA of oxidatively-stressed and cancer cells

氧化应激细胞和癌细胞 DNA 中核糖核苷酸的核糖测序谱和分析

基本信息

批准号：
9921385
负责人：
Francesca Storici
金额：
$ 27.63万
依托单位：
GEORGIA INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-08-01 至 2022-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9921385
关键词：
Antimycin A Base Excision Repairs Biological Assay Biological Markers Cancer Biology Cancer Etiology Cancer cell line Cell Line Cells Cleaved cell Comprehension DNA DNA Damage DNA Maintenance DNA Repair DNA Repair Gene DNA Repair Pathway DNA biosynthesis DNA sequencing DNA-Directed DNA Polymerase Data Defect Deoxyribonucleotides Deoxyribose Detection Development Distal Embryo Excision Excision Repair Exposure to Fibroblasts Genetic Transcription Genome Stability Genomic DNA Genomic Instability Genomics Hela Cells Human Hydrogen Peroxide In Vitro Investigation Laboratories Libraries Link Liver Lyase Malignant Neoplasms Malignant neoplasm of liver Maps Methods Mismatch Repair Mitochondria Mitochondrial DNA Modeling Mus Mutation Mutation Spectra Nature Normal Cell Normal tissue morphology Nuclear Nucleotide Excision Repair Nucleotides Oxidation-Reduction Oxidative Stress Oxides Paraquat Pathway interactions Phenotype Physiological Primary carcinoma of the liver cells Process RNA Reactive Oxygen Species Regulation Reproducibility Ribonucleases Ribonucleosides Ribonucleotides Ribose Role Rotenone Saccharomyces cerevisiae Saccharomycetales Sampling Site Stress Superoxide Dismutase Techniques Technology Testing Time Tissues Variant Yeasts cancer cell catalase design endonuclease environmental stressor genome integrity hepatocellular carcinoma cell line inhibitor/antagonist insight interest knock-down mouse genome mutant preservation reconstitution reference genome repair enzyme repaired stressor sugar superoxide dismutase 1 tool toxicant tumor yeast genome

项目摘要

Project Summary Ribonucleoside monophosphates (rNMPs), the subunits of RNA, are the most common non-canonical nucleotides found in genomic DNA. Inactivation of ribonuclease (RNase) H2, which is the major player in the removal of rNMPs from nuclear DNA (nDNA), allowed detection of over one million rNMPs in the mouse genome and ~2,400 rNMPs in the budding yeast genome. rNMPs distort the DNA double helix, modulating or altering DNA functions and increasing DNA fragility and instability. There is a need to determine where rNMP sites are in DNA, especially in cells with abnormal genome stability, like cancer cells. We recently developed a method, ribose-seq, to map rNMPs present in genomic DNA (Koh et al., Nature Methods, 2015). We applied ribose-seq to yeast Saccharomyces cerevisiae RNase H2 deficient cells, and we revealed widespread but not random distribution of rNMPs with several hotspots in nDNA and mitochondrial DNA (mtDNA). A proven, though poorly explored, cause of rNMP inclusion in DNA is oxidative stress, which, through reactive oxygen species (ROS), converts deoxyribose to ribose both in the deoxyribonucleotide pool and within DNA. Moreover, ROS not only produce abasic DNA, which is repaired via the base excision repair (BER) pathway, but also abasic RNA. Because we recently demonstrated that the BER apurinic/apyrimidinic endonuclease Ape1 cleaves also abasic RNA, we aim to determine if BER is involved in removal of rNMPs from DNA. Currently, it is unknown whether and how the profile of rNMP incorporation in genomic DNA changes upon oxidative stress, and whether there is any link with cancer phenotype. Are there genomic sites (i.e. transcriptionally active regions) that are more prone to rNMP formation upon exposure to ROS? Is there a correlation between rNMP and mutation sites occurring in oxidatively stressed and/or cancer cells? In Aim 1, applying ribose-seq, we will reveal for the first time, the spectrum of rNMP incorporation in different conditions of oxidative stress in nDNA and mtDNA of S. cerevisiae RNase H2-deficient cells. The rNMP profiles will be analyzed and compared with those of the same yeast cells not exposed to the oxidative stressors, and also with mutation spectra of the same ROS-exposed cells. Because RNase H2 activity for rNMP removal was not found in mitochondria, mtDNA could be particularly sensitive to rNMP incorporation during oxidative stress. Thus, in Aim 2 we will perform profile and analysis of rNMPs in mtDNA of yeast and normal mammalian RNase H2-proficient cells exposed to oxidative stress and sensitized to it by using mutants and inhibitors of BER factors. rNMP maps will be also compared with mutation maps. In Aim 3, we will perform profile and analysis of rNMPs in mtDNA of cancer cells. Cancer cells from different human hepatic cancer cell lines, from a selection of human bioptic hepatocarcinoma samples (tumoral and distal liver tissues) and HeLa cells reconstituted with different functional variants of Ape1, will be processed to obtain purified mtDNA, which will be analyzed for rNMP distribution and hotspots of incorporation to identify significant biomarkers.

项目概要单磷酸核糖核苷 (rNMP) 是 RNA 的亚基，是最常见的非典型物质基因组 DNA 中发现的核苷酸。核糖核酸酶 (RNase) H2 失活，这是从核 DNA (nDNA) 中去除 rNMP，从而可以在小鼠体内检测到超过 100 万个 rNMP 基因组和芽殖酵母基因组中约 2,400 个 rNMP。 rNMPs 扭曲 DNA 双螺旋，调节或改变 DNA 功能并增加 DNA 脆弱性和不稳定性。需要确定rNMP在哪里位点位于 DNA 中，尤其是在基因组稳定性异常的细胞中，例如癌细胞。我们最近开发了一个核糖测序方法可对基因组 DNA 中存在的 rNMP 进行定位（Koh 等人，NatureMethods，2015）。我们申请了对酿酒酵母 RNase H2 缺陷细胞进行核糖测序，我们发现广泛存在但并非如此 rNMP 的随机分布，在 nDNA 和线粒体 DNA (mtDNA) 中具有多个热点。 rNMP 包含在 DNA 中的一个已被证实的原因是氧化应激，尽管对此研究还很少。活性氧 (ROS)，将脱氧核糖核苷酸池中和内部的脱氧核糖转化为核糖脱氧核糖核酸。此外，ROS不仅产生脱碱基DNA，通过碱基切除修复（BER）进行修复途径，还有脱碱基RNA。因为我们最近证明了 BER 无嘌呤/无嘧啶核酸内切酶 Ape1 也切割脱碱基 RNA，我们的目的是确定 BER 是否参与 rNMP 的去除来自DNA。目前，尚不清楚 rNMP 是否以及如何掺入基因组 DNA 中。氧化应激引起的变化，以及是否与癌症表型有任何联系。是否有基因组位点（即转录活性区域）在暴露于 ROS 时更容易形成 rNMP？有没有一个 rNMP 与氧化应激和/或癌细胞中发生的突变位点之间的相关性？在目标 1 中，应用核糖测序，我们将首次揭示 rNMP 掺入谱酿酒酵母 RNase H2 缺陷细胞的 nDNA 和 mtDNA 氧化应激的不同条件。这 rNMP 谱将被分析并与未暴露于氧化的相同酵母细胞的谱进行比较。应激源，以及相同 ROS 暴露细胞的突变谱。因为 RNase H2 活性为线粒体中未发现 rNMP 去除，mtDNA 可能对 rNMP 掺入特别敏感在氧化应激期间。因此，在目标 2 中，我们将对酵母和线粒体 DNA 中的 rNMP 进行概况分析和分析。正常哺乳动物 RNase H2 熟练细胞暴露于氧化应激并通过使用突变体对其敏感和 BER 因素的抑制剂。 rNMP 图谱还将与突变图谱进行比较。在目标 3 中，我们将执行癌细胞 mtDNA 中 rNMP 的概况和分析。来自不同人类肝癌细胞的癌细胞细胞系，来自精选的人类活检肝癌样本（肿瘤和远端肝组织）和 HeLa 用 Ape1 的不同功能变体重组的细胞将被处理以获得纯化的 mtDNA，将分析 rNMP 分布和掺入热点，以确定重要的生物标志物。

项目成果

期刊论文数量（10）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Frequency and patterns of ribonucleotide incorporation around autonomously replicating sequences in yeast reveal the division of labor of replicative DNA polymerases.

DOI：
10.1093/nar/gkab801
发表时间：
2021-10-11
期刊：
Nucleic acids research
影响因子：
14.9
作者：
Xu P;Storici F
通讯作者：
Storici F

Abasic and oxidized ribonucleotides embedded in DNA are processed by human APE1 and not by RNase H2.

DOI：
10.1093/nar/gkx723
发表时间：
2017-11-02
期刊：
Nucleic acids research
影响因子：
14.9
作者：
Malfatti MC;Balachander S;Antoniali G;Koh KD;Saint-Pierre C;Gasparutto D;Chon H;Crouch RJ;Storici F;Tell G
通讯作者：
Tell G

Mapping ribonucleotides embedded in genomic DNA to single-nucleotide resolution using Ribose-Map.

DOI：
10.1038/s41596-021-00553-x
发表时间：
2021-07
期刊：
NATURE PROTOCOLS
影响因子：
14.8
作者：
Gombolay, Alli L.;Storici, Francesca
通讯作者：
Storici, Francesca

Addendum: Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA.

附录：核糖测序：嵌入基因组 DNA 中的核糖核苷酸的全局作图。

DOI：
10.1038/s41592-019-0505-9
发表时间：
2019
期刊：
Nature methods
影响因子：
48
作者：
Koh,KyungDuk;Balachander,Sathya;Hesselberth,JayR;Storici,Francesca
通讯作者：
Storici,Francesca

Capture of Ribonucleotides in Yeast Genomic DNA Using Ribose-Seq.

使用核糖测序捕获酵母基因组 DNA 中的核糖核苷酸。

DOI：
10.1007/978-1-4939-9736-7_2
发表时间：
2019
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
0
作者：
Balachander,Sathya;Yang,Taehwan;Newnam,Gary;El-Sayed,WaleedMM;Koh,KyungDuk;Storici,Francesca
通讯作者：
Storici,Francesca