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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.

项目总结

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

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

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

Ribose-Map: a bioinformatics toolkit to map ribonucleotides embedded in genomic DNA.