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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：
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（由申请人提供）：内源性（例如氧自由基）对 DNA 的持续损伤主要通过碱基切除 DNA 修复（BER）途径来抵消。通过体外方法已经获得了关于这一过程的大量知识，并且遗传学研究支持关键蛋白质（例如 Ape1 脱碱基核酸内切酶）在哺乳动物细胞中的重要作用。 BER 子途径已被描述为指导“长补丁”(LP) BER 中单个核苷酸或多个核苷酸的替换。后者对于氧化脱碱基位点 2-脱氧核糖内酯尤其重要，但可能还有其他需要 LP-BER 的损伤。缺少的是一种稳健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通路分布将阐明不同细胞类型和不同细胞的遗传稳定性机制。 DNA 损伤。我们建议开发一种解决这个问题的新方法，通过建立一种使用掺入质粒底物中的质量标记核苷酸进行转染至哺乳动物细胞的测定方法。在这种方法中，目标病变的 3' 下游（或周围）核苷酸用 13C 或 15N 标记，并置于非复制质粒载体中用于转染到哺乳动物细胞中。体内修复将取代“重”核苷酸，其长度可以通过回收 DNA 后的后续质谱测定来确定。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将促进这些过程。有两个具体目标： 1. 我们之前用于在体外证明 2-脱氧核糖内酯 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）途径的损伤平台，通过包含周围质量标记的合成寡核苷酸插入核苷酸。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测试，后者预计主要显示 LP-BER。 2. 将载体转染到正常和 POLB 缺陷的细胞中，以评估 BER 子通路作用于这些（以及最终其他）DNA 损伤的体内贡献。