MAPPING DNA BINDING SITES IN PROTEINS BY UV CROSSLINKING & MASS SPECTROMETRY

通过 UV 交联绘制蛋白质中的 DNA 结合位点

• 批准号: 6308903
• 负责人: MARTIN D SHETLAR
• 金额: $ 0.99万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2002-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6308903
• 关键词: biological products; biomedical resource; neoplasm /cancer; nucleic acids; proteins; spectrometry; structural biology; technology /technique

This study aims to establish and exploit a new method of determining the location of DNA binding sites in proteins at the level of individual amino acids. The method is potentially applicable to any protein that binds non-covalently to DNA. UV light can induce protein-DNA crosslinking in cells and protein that binds to DNA has a much greater tendency to become crosslinked than unbound protein. Specifically, that portion of the protein that is in contact with the DNA will be crosslinked. Thus, UV light can be used as a "zero-length" crosslinking reagent. Knowledge of the contact regions between DNA and protein in DNA-protein complexes is an essential prerequisite for achieving an understanding of biological processes at the molecular level. Proteins bind to DNA for a variety of essential purposes, as well as being implicated in cancer, as for example, the gene products of tumor suppressor genes and oncogenes. Two types of processes relevant to cancer research are transcriptional regulation and DNA repair. The two mainobstacles to the study of these crosslinked DNA-protein systems have been the difficulty of analyzing for the modified amino acids in the protein, and the lack of knowledge about the stability of the crosslinks formed. In order to overcome these obstacles, we propose to use molecular weight determination and sequencing by tandem mass spectrometry to perform the necessary peptide analyses. Peptides to be analyzed will be produced from the protein-DNA complex by enzyme digestion of the protein and the attached DNA as necessary. Initially, we intend to determine the exact amino acid residues in the rat DNA polymerase b protein that cross-link to DNA when exposed to UV light. We will also prepare and characterize peptide-nucleobase, peptide-oligonucleotide, and protein-oligonucleotide crosslinked species. Our initial goals are: 1) To establish that UV photoinduced crosslinking occurs between the amino acid residues and the nucleic acid bases at specific binding site(s) in the polymerase b protein. 2) To isolate and analyze crosslinked peptides by means of mass spectrometry. Model peptides and oligonuleotides will be used in the initial stages of this work. 3) To determine the optimum experimental conditions and procedures for production, separation, and analysis of crosslinked proteins and to establish reasonable minimum sample requirements.

这项研究旨在建立和利用一种新的方法 确定DNA结合位点在蛋白质上的位置 单个氨基酸。 该方法可能适用于 任何与DNA无共价结合的蛋白质。 紫外线可以诱导 与DNA结合的细胞和蛋白质中的蛋白-DNA交联具有 与未结合的蛋白质交联的趋势要大得多。 具体而言，与之接触的蛋白质的一部分 DNA将交联。 因此，紫外线可以用作 “零长度”交联试剂。 了解接触区域 DNA和DNA蛋白质复合物中的蛋白质之间是必不可少的 实现对生物过程的理解的先决条件 分子水平。 蛋白质与DNA结合多种必需 目的以及与癌症有关的目的，例如 肿瘤抑制基因和癌基因的基因产物。 两种类型 与癌症研究相关的过程是转录调节 和DNA修复。 研究这些研究 交联的DNA-蛋白系统一直很难分析 对于蛋白质中的修饰氨基酸，缺乏知识 关于形成的交联的稳定性。 为了克服 这些障碍，我们建议使用分子量测定和 通过串联质谱进行测序以执行必要 肽分析。 要分析的肽将由 蛋白质蛋白DNA复合物通过蛋白质的酶消化和 必要时附着的DNA。 最初，我们打算确定 大鼠DNA聚合酶B蛋白中的确切氨基酸残基 暴露于紫外线时，交联至DNA。 我们还将准备和 表征肽核碱基，肽 - 寡核苷酸和 蛋白 - 寡核苷酸交联物种。 我们的最初目标是： 1）确定紫外线诱导的交联发生在 氨基酸残基和特异性结合处的核酸碱基 聚合酶B蛋白中的位点。 2）分离和分析 通过质谱法交联的肽。 模型肽 和寡核苷酸将在本工作的初始阶段使用。 3）确定最佳的实验条件和程序 交联蛋白的生产，分离和分析以及 确定合理的最低样本要求。