Single- and multinucleotide base excision DNA repair pathways in vivo

体内单核苷酸和多核苷酸碱基切除 DNA 修复途径

基本信息

批准号：
8959001
负责人：
Bruce F. Demple
金额：
$ 20.41万
依托单位：
STATE UNIVERSITY NEW YORK STONY BROOK
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-08-01 至 2017-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8959001
关键词：
5&apos -deoxyribose phosphate lyase Affinity Affinity Chromatography Base Excision Repairs Biological Assay Breast Cancer Cell Cell Cycle Cell Extracts Cell Maintenance Cell-Free System Cells DNA DNA Polymerase beta DNA Repair Pathway DNA glycosylase DNA lesion DNA-(apurinic or apyrimidinic site) lyase DNA-Directed DNA Polymerase DNA-protein crosslink Deamination Dependence Development Digestion Embryo Enzymes Excision Exonuclease Fibroblasts Generations Genetic Genetic study Human In Vitro Incubated Individual Knowledge Label Lesion Life MALDI-TOF Mass Spectrometry Malignant Neoplasms Mammalian Cell Mass Spectrum Analysis Metabolic Mouse Cell Line Mus Nucleotide Excision Repair Nucleotides Oligonucleotides Organ POLB gene Pathway interactions Phase Plasmid Cloning Vector Plasmids Polymerase Principal Investigator Process Proteins Reaction Reactive Oxygen Species Recombinants Recovery Regimen Relative (related person)Residual state Resistance Role Site Source Testing Time Transfection Uracil Uracil Nucleotides Variant analog base cancer cell cancer therapy cell type endonuclease in vivo method development mutant neoplastic cell novel strategies oxidation personalized medicine plasmid DNA prevent programs public health relevance repaired resistance mechanism response restriction enzyme tetrahydrofuran tumor uracil-DNA glycosylase vector

项目摘要

DESCRIPTION (provided by applicant): The incessant damage to DNA from endogenous sources (e.g., oxygen radicals) is counteracted mainly by the base excision DNA repair (BER) pathway. Considerable knowledge of this process has been obtained by in vitro approaches, and genetic studies support important roles for key proteins such as the Ape1 abasic endonuclease in mammalian cells. BER sub-pathways have been described that direct the replacement of a single-nucleotide, or of several nucleotides in "long-patch" (LP) BER. The latter is especially important for the oxidized abasic site 2-deoxyribonolactone, but there are probably other lesions requiring LP-BER. What is missing is a robust and precise approach to characterizing these pathways in living cells. Understanding the actual pathway distribution in intact cells will illuminate genetic stability mechanisms in different cell types and for different DNA lesions. We propose to develop a novel approach to this problem, by establishing an assay using mass-labeled nucleotides incorporated in plasmid substrates for transfection into mammalian cells. In this approach, the target lesion will have the 3' downstream (or surrounding) nucleotides labeled with 13C or 15N, and placed in a non-replicating plasmid vector for transfection into mammalian cells. Repair in vivo will replace "heavy" nucleotides, the extend of which can be determined by subsequent mass spectrometry after recovery of the DNA. These processes will be facilitated by adjacent restriction enzyme sites and the presence of biotinylated nucleotides for affinity purification. There are two specific aims: 1. A plasmid vector we previously used to demonstrate LP-BER of 2-deoxyribonolactone in vitro will be used as a platform for lesions that delineate the single-nucleotide (uracil) and LP-BER (the stable abasic analog tetrahydrofuran) pathways, inserted via synthetic oligonucleotides containing surrounding mass-labeled nucleotides. These substrates will be tested initially in extracts from normal and DNA polymerase beta- deficient cells, with the latter expected to display predominantly LP-BER. 2. The vectors will be transfected into normal and POLB-deficient cells to assess the in vivo contribution of the BER sub-pathways acting on these (and eventually other) DNA lesions.

描述（由适用提供）：内源性来源（例如氧自由基）对DNA的不断损害主要由基本惊喜DNA修复（BER）途径来抵消。通过体外方法获得了有关此过程的广泛了解，遗传研究支持关键蛋白（例如APE1脓肿性核酸内切酶）在哺乳动物细胞中的重要作用。已经描述了指导替换单核苷酸或“长点”（LP）BER中的几个核苷酸的ber子街道。后者对于氧化的缺失位点2-脱氧核酸酮酮尤其重要，但是可能还有其他需要LP-ber的病变。缺少的是一种表征活细胞中这些途径的强大而精确的方法。了解完整细胞中的实际途径分布将阐明不同细胞类型中的遗传稳定性机制，并且 DNA病变。我们建议通过在质粒底物中纳入质量标记的核苷酸来建立一种新的方法来开发这种问题的新方法，以转化为哺乳动物细胞。在这种方法中，靶病变将具有标记为13C或15N标记的下游（或周围）核苷酸的3'（或周围），并将其放置在非复制质粒载体中，以转化为哺乳动物细胞。体内修复将取代“重”核苷酸，这些过程的延伸将通过恢复DNA后随后的质谱法确定。这些过程将通过相邻限制酶位点和生物素基化的核苷酸来制备以进行亲和力纯化。有两个具体的目的：1。我们先前用来在体外用来证明2-脱氧核酸酮的LP-Ber将用作鉴定单核苷酸（Uracil）（Uracil）和LP-ber（稳定的抗甲基甲基甲虫）核酸酯的核酸酯的病变的平台。这些底物最初将在正常和DNA聚合酶β缺陷型细胞的提取物中进行测试，后者预计将主要显示出LP-ber。 2。载体将被翻译成正常和POLB缺陷的细胞，以评估作用于这些（甚至其他）DNA病变的BER子座管的体内贡献。