SINGLE-STRANDED NUCLEIC ACID BINDING PROTEINS

单链核酸结合蛋白

• 批准号: 3292953
• 负责人: Kenneth Robert WILLIAMS
• 金额: $ 20.66万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-03-01 至 1993-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3292953
• 关键词: DNA; DNA binding protein; DNA directed DNA polymerase; DNA replication; Escherichia coli; X ray crystallography; aminoacid; bacteriophage T4; binding proteins; chemical binding; conformation; crosslink; genetic regulation; genetic transcription; high performance liquid chromatography; mouse sarcoma virus; nuclear magnetic resonance spectroscopy; nuclease; nucleic acid structure; phenylalanine; protein engineering; protein structure function; synthetic peptide; temperature sensitive mutant; tyrosine; zinc

The single-stranded DNA binding proteins encoded by gene 32 of bacteriophage T4 (gp32) and ssb of E. coli (SSB) have served as prototypes for a class of proteins required in stoichiometric amounts in DNA replication, repair and recombination. In general, binding of these proteins imposes a conformation onto the ssDNA that is both resistant to nucleases and optimum to then serve as a substrate for other enzymes, such as DNA polymerases, involved in nucleic acid metabolism. Our research increasingly suggests that gp32 and SSB share many features in common with functionally unrelated eukaryotic nucleic acid binding proteins. Hence the function of the acidic COOH-terminus of these proteins appears analogous to the corresponding region in high mobility group proteins; as in the case of gp32 and SSB, hydrophobic interactions involving aromatic amino acid residues have also been implicated in the binding of heterogeneous nuclear RNA binding proteins. Finally, the zinc binding domain of gp32 appears to represent a nucleic acid binding motif shared by eukaryotic transcription factors as well as nucleic acid binding proteins encoded by murine leukemia and acquired immune deficiency viruses. To continue this line of research, we propose to more clearly define the role of the zinc ion in gp32 and to use an in vitro mutagenesis/1H-NMR/physicochemical approach to identify amino acids in gp32 and SSB that are directly involved in ssDNA binding. High priority will be given towards crystallizing gp32. Two new conditional lethal mutations in SSB will be characterized and limited proteolysis as well as photocross-linking experiments will be undertaken to determine how the tetrameric structure of SSB relates to ssDNA binding. A novel approach using a thermolabile SSB mutant and peptide competition experiments is planned to identify amino acids essential for SSB tetramer formation. This research will culminate in the synthesis and characterization of peptides corresponding to presumed functional domains in SSB and gp32 as well as to other related nucleic acid binding proteins. The structures of these synthetic peptides will then be varied in such a way as to test our ideas concerning the relationship of structure and function in nucleic acid binding proteins. These proposed studies are basic to our understanding of those physiological processes such as DNA replication, transcription and translation that require a single-stranded nucleic acid template.

由基因32编码的单链DNA结合蛋白， 噬菌体T4（gp 32）和E.大肠杆菌（SSB）已作为 化学计量中所需的一类蛋白质的原型 大量的DNA复制、修复和重组。 在 一般来说，这些蛋白质的结合将构象强加给 对核酸酶具有抗性并且对核酸酶具有最佳抗性的ssDNA 作为其他酶的底物，如DNA聚合酶， 参与核酸代谢。 我们的研究越来越多 这表明gp 32和SSB有许多共同的特征， 功能无关的真核核酸结合蛋白。 因此，这些化合物的酸性COOH-末端的功能 蛋白质似乎类似于高水平的相应区域， 迁移族蛋白;如在gp 32和SSB的情况下， 涉及芳香族氨基酸残基的疏水相互作用 也与异质核的结合有关， RNA结合蛋白。 最后，gp 32的锌结合结构域 似乎代表了一个核酸结合基序， 真核转录因子以及核酸结合 小鼠白血病和获得性免疫 缺陷病毒 为了继续这一研究方向，我们建议更明确地 确定锌离子在gp 32中的作用， 诱变/1H-NMR/物理化学方法鉴定 gp 32和SSB中直接参与ssDNA的氨基酸 约束力 gp 32的结晶化将被给予高度优先考虑。 SSB中两个新的条件性致死突变将被表征 以及有限的蛋白质水解和光交联实验 将进行，以确定如何四聚体结构的 SSB涉及ssDNA结合。 一种新颖的方法， 热不稳定SSB突变体和肽竞争实验是 计划鉴定SSB四聚体必需的氨基酸 阵 这项研究将最终在合成和 对应于假定功能的肽的表征 SSB和gp 32中的结构域以及其它相关核酸 结合蛋白 这些合成肽的结构将 然后以这样的方式来测试我们的想法， 核酸结合的结构与功能关系 proteins. 这些拟议中的研究是我们理解 这些生理过程，如DNA复制， 转录和翻译需要单链的 核酸模板。