DNA-Binding Protein Discovery by the Combinatorial Method REPSA

描述(由申请人提供)：我们的目标是通过新的组合方法，限制内切酶保护，选择和扩增(REPSA)来鉴定它们的首选结合部位，从而提高我们对预测和/或意外DNA结合蛋白的理解。虽然人类基因组计划已经为人类基因组和其他模式生物提供了丰富的信息，但关于单个基因及其编码产物所起的生物学作用仍有许多有待确定的地方。例如，虽然细菌大肠杆菌K12菌株有4364个开放阅读框，但只有大约一半的基因通过遗传、生化或分子生物学手段得到了很好的表征。许多已知的基因(260+)编码的蛋白质可能与特定的DNA序列结合。然而，对于这些蛋白质中的大多数(&gt；200)，它们首选的DNA结合位点还没有得到经验上的确定。我们开发了一种组合方法REPSA，它不需要任何事先知道的配体来确定其在双链DNA上的首选结合位置。因此，我们假设REPSA可以用来确定模式生物E.ColiK-12中未鉴定蛋白的首选DNA结合位点。我们提出了以下四个具体目标：(1)使用REPSA识别首选的DNA结合位点，输入范围从纯化的大肠杆菌蛋白到细菌提取物，大规模并行DNA测序和生物信息学分析(例如，基序的多重期望最大值，Meme)。(2)必要时，通过DNA亲和/磁珠捕获和多肽指纹图谱识别结合到首选DNA结合部位的蛋白质。(3)用重组蛋白、点突变寡核苷酸和荧光偏振结合试验验证蛋白质与DNA的结合并确定结合的特异性。(4)通过在现有数据库(如EcoCyc、EcoliWiki、Ecogene、RegulonDB、UniProtKB)中寻找单个基序(FIMO)和注释，在大肠杆菌基因组上定位这些蛋白质及其结合部位，从而提出这些蛋白质及其结合部位的潜在生物学功能。我们希望我们的研究最终将促进我们和其他实验室的大量研究，从而在分子水平上更好地理解大肠杆菌生物学，并为其他生物的类似研究提供一个框架。

DESCRIPTION (provided by applicant): Our goal is to advance our understanding of predicted and/or unexpected DNA-binding proteins through the identification of their preferred binding sites by the novel combinatorial approach, Restriction Endonuclease Protection, Selection and Amplification (REPSA). While the Human Genome Project has yielded a wealth of information for both the human genome and other model organisms, much remains to be determined regarding individual genes and the biological roles played by their encoded products. For example, while the bacteria Escherichia coli strain K12 has 4364 open reading frames, only about half of these genes have been well characterized by genetic, biochemical or molecular biological means. Many of the known genes (260+) encode for proteins that presumably bind specific DNA sequences. However, for most of these proteins (>200) their preferred DNA-binding sites have not been determined empirically. We have developed a combinatorial approach, REPSA, which does not require any prior knowledge of a ligand in order to determine its preferred binding site on duplex DNA. Thus we hypothesize that REPSA can be used to identify the preferred DNA- binding sites of uncharacterized proteins in the model organism E. coli K-12. We propose the following four Specific Aims: (1) Identify preferred DNA-binding sites using REPSA with inputs ranging from purified E. coli proteins to bacterial extracts, massively parallel DNA sequencing, and bioinformatics analyses (e.g., Multiple Expectation Maximum for Motif Elicitation, MEME). (2) When necessary, identify proteins bound to identify preferred DNA-binding sites by DNA- affinity/magnetic bead capture and peptide fingerprinting. (3) Validate protein-DNA binding and determine binding specificity using recombinant proteins, point-mutated oligonucleotides, and fluorescence polarization binding assays. (4) Propose potential biological functions for these proteins and their binding sites through binding site mapping on the E. coli genome using Find Individual Motif Occurrence (FIMO) and annotations in available databases (e.g., EcoCyc, EcoliWiki, EcoGene, RegulonDB, UniProtKB). We expect our research to ultimately catalyze numerous studies by us and other laboratories leading to a better understanding of E. coli biology at a molecular level and provide a framework for similar studies in other organisms.