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

本研究旨在建立和开发一种新的方法。 在蛋白质水平上确定DNA结合位点的位置 单独的氨基酸组成。该方法具有潜在的适用性。 以非共价方式与DNA结合的任何蛋白质。紫外光可以诱导 细胞中的蛋白质-DNA交联和与DNA结合的蛋白质具有 与未结合的蛋白质相比，更容易发生交联化。 具体地说，与蛋白质接触的那部分蛋白质 DNA将会被交联。因此，紫外光可以作为一种 “零长度”交联剂。接触区域的知识 DNA和蛋白质之间的DNA-蛋白质复合体是必不可少的 实现对生物过程的了解的先决条件 分子水平。蛋白质与DNA结合，以获得各种必需的 目的，以及与癌症有关，例如， 抑癌基因和癌基因的基因产物。两种类型的 与癌症研究相关的过程是转录调控 和DNA修复。研究这些问题的两个主要障碍 DNA-蛋白质交联体系一直是分析的难点 对于蛋白质中的修饰氨基酸，以及缺乏知识 关于形成的交联物的稳定性。为了克服这个问题 对于这些障碍，我们建议使用分子量测定和 用串联质谱仪进行测序以执行必要的 多肽分析。要分析的多肽将从 蛋白质-DNA复合体的蛋白质和DNA的酶消化 根据需要附上DNA。最初，我们打算确定 大鼠DNA聚合酶b蛋白中准确的氨基酸残基 当暴露在紫外光下时，会与DNA发生交叉连接。我们还将准备和 表征多肽-碱基、多肽-寡核苷酸和 蛋白质-寡核苷酸交联物种。我们最初的目标是： 1)确定紫外光诱导的交联剂发生在 氨基酸残基与特定结合的核酸碱基 聚合酶b蛋白中的一个位点(S)。2)分离分析 通过质谱学手段进行交联肽的研究。模型肽 而寡核苷酸将用于这项工作的初始阶段。 3)确定最佳实验条件和程序 交联蛋白和TO的生产、分离和分析 制定合理的最低样品要求。

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.