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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：
9115558
负责人：
Bruce F. Demple
金额：
$ 16.97万
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/9115558
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 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（由申请人提供）：内源性来源（例如，氧自由基）主要通过碱基切除DNA修复（BER）途径抵消。这一过程的相当多的知识已经获得了体外方法，遗传研究支持的重要作用，如哺乳动物细胞中的Ape 1无碱基核酸内切酶的关键蛋白质。已经描述了BER子途径，其指导“长补丁”（LP）BER中的单个核苷酸或几个核苷酸的替换。后者对于氧化脱碱基位点2-脱氧核糖内酯尤其重要，但可能还有其他病变需要LP-BER。缺少的是一种强大而精确的方法来表征活细胞中的这些途径。了解完整细胞中的实际途径分布将阐明不同细胞类型和不同细胞类型中的遗传稳定性机制。 DNA损伤我们建议开发一种新的方法来解决这个问题，通过建立一个测定使用质量标记的核苷酸纳入质粒基板转染到哺乳动物细胞。在该方法中，靶病变将具有用13 C或15 N标记的3'下游（或周围）核苷酸，并置于非复制型质粒载体中用于转染到哺乳动物细胞中。体内修复将替换“重”核苷酸，其程度可以在DNA回收后通过随后的质谱法确定。这些过程将通过相邻的限制酶位点和用于亲和纯化的生物素化核苷酸的存在来促进。有两个具体目标：1。我们先前用于体外证明2-脱氧核糖内酯的LP-BER的质粒载体将用作描绘单核苷酸（尿嘧啶）和LP-BER（稳定的无碱基类似物四氢呋喃）途径的病变的平台，通过含有周围质量标记核苷酸的合成寡核苷酸插入。这些底物最初将在正常细胞和DNA聚合酶β缺陷细胞的提取物中进行检测，预计后者主要显示LP-BER。2.将载体转染到正常和POLB缺陷细胞中，以评估BER子途径对这些（以及最终其他）DNA损伤的体内作用。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Maintenance of Flap Endonucleases for Long-Patch Base Excision DNA Repair in Mouse Muscle and Neuronal Cells Differentiated In Vitro.

DOI：
10.3390/ijms241612715
发表时间：
2023-08-12
期刊：
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
影响因子：
5.6
作者：
Caston, Rachel A.;Fortini, Paola;Chen, Kevin;Bauer, Jack;Dogliotti, Eugenia;Yin, Y. Whitney;Demple, Bruce
通讯作者：
Demple, Bruce

Risky repair: DNA-protein crosslinks formed by mitochondrial base excision DNA repair enzymes acting on free radical lesions.