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

项目摘要 核糖核苷一磷酸(RNMPs)是核糖核酸的亚基，是最常见的非正则 在基因组DNA中发现的核苷酸。核糖核酸酶(RNase)H2的失活，它是 从核DNA(NDNA)中去除rNMPs，允许在小鼠中检测到100多万rNMPs 芽殖酵母基因组中含有约2,400个rNMPs。RNMP扭曲DNA双螺旋，调节或 改变DNA功能，增加DNA的脆弱性和不稳定性。需要确定rNMP在哪里 位点在DNA中，特别是在基因组稳定性异常的细胞中，如癌细胞。我们最近开发了一种 方法，核糖-序列，以定位基因组DNA中存在的rNMP(Koh等人，自然方法，2015)。我们申请了 核糖序列到酵母RNaseH2缺陷的酵母细胞，我们揭示了广泛的但不是 随机分布的rNMPs在nDNA和线粒体DNA(MtDNA)中有几个热点。 DNA中包含rNMP的一个已证实的原因是氧化应激，通过 活性氧物种(ROS)，在脱氧核糖核酸库和内都能将脱氧核糖转化为核糖 DNA此外，ROS不仅产生通过碱基切除修复(BER)修复的基本DNA 途径，但也有基本的RNA。因为我们最近证明了BER无嘌呤/无嘧啶 核酸内切酶APE1也切割基本的RNA，我们的目标是确定BER是否参与rNMPs的去除 从DNA中。目前还不清楚rNMP在基因组dna中的掺入情况。 氧化应激的变化，以及是否与癌症表型有任何联系。有没有基因组位点？ (即转录活性区域)在暴露于ROS时更容易形成rNMP？有没有 氧化应激和/或癌细胞中rNMP与突变位点的相关性？ 在目标1中，应用核糖序列，我们将首次揭示rNMP掺入的光谱。 酿酒酵母核糖核酸酶H2缺陷细胞nDNA和mtDNA氧化应激的不同条件。这个 将分析rNMP图谱，并将其与未暴露在氧化环境中的相同酵母细胞的rNMP图谱进行比较 应激源，以及同样暴露在ROS下的细胞的突变谱。因为RNaseH2的活性 线粒体中没有发现rNMP的去除，mtDNA对rNMP的掺入特别敏感 在氧化应激过程中。因此，在目标2中，我们将对酵母mtDNA中的rNMPs进行图谱和分析 正常哺乳动物RNaseH2熟练细胞暴露于氧化应激并通过突变体致敏 和误码率因子的抑制剂。RNMP图谱也将与突变图谱进行比较。在目标3中，我们将表演 癌细胞线粒体DNA中rNMPs的图谱及分析。不同人肝癌细胞来源的癌细胞 取自一组人肝癌标本(肿瘤和远端肝组织)和HeLa 用APE1不同功能变体重组的细胞将被处理以获得纯化的mtDNA，该mtDNA 将分析rNMP的分布和掺入热点，以确定重要的生物标志物。

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.