SINGLE-STRANDED NUCLEIC ACID BINDING PROTEINS

单链核酸结合蛋白

• 批准号: 3292952
• 负责人: Kenneth Robert WILLIAMS
• 金额: $ 17.13万
• 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/3292952
• 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 (gp32) 和大肠杆菌的 SSB (SSB) 已作为 化学计量所需的一类蛋白质的原型 DNA 复制、修复和重组的量。 在 一般来说，这些蛋白质的结合将构象强加于 既能抵抗核酸酶又能优化核酸酶的单链DNA 作为其他酶的底物，例如 DNA 聚合酶， 参与核酸代谢。 我们的研究日益 表明 gp32 和 SSB 有许多共同点 功能无关的真核核酸结合蛋白。 因此，这些的酸性COOH末端的功能 蛋白质似乎与高水平的相应区域相似 移动基团蛋白；与 gp32 和 SSB 的情况一样， 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，gp32 的锌结合域 似乎代表了共享的核酸结合基序 真核转录因子以及核酸结合 小鼠白血病和获得性免疫编码的蛋白质 缺陷病毒。 为了继续这一研究方向，我们建议更明确地 定义 gp32 中锌离子的作用并使用体外 诱变/1H-NMR/理化方法鉴定 gp32 和 SSB 中直接参与 ssDNA 的氨基酸 绑定。 将高度优先考虑 gp32 的结晶。 SSB 中两种新的条件致死突变将得到表征 和有限的蛋白水解以及光交联实验 将进行以确定四聚体结构如何 SSB 与 ssDNA 结合有关。 一种新颖的方法使用 不耐热的 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 形成。 这项研究最终将在合成和 对应于假定功能的肽的表征 SSB 和 gp32 以及其他相关核酸中的结构域 结合蛋白。 这些合成肽的结构将 然后以这样的方式进行变化，以测试我们关于 核酸结合中结构与功能的关系 蛋白质。 这些拟议的研究是我们理解的基础 那些生理过程，例如DNA复制， 需要单链的转录和翻译 核酸模板。