A.T-SPECIFIC DNA-BINDING PROPERTIES OF A HUMAN PROTEIN

人类蛋白质的 A.T 特异性 DNA 结合特性

• 批准号: 3305771
• 负责人: RAYMOND REEVES
• 金额: $ 15.63万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 1994-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3305771
• 关键词: DNA binding protein; DNA footprinting; cell cycle; chimeric proteins; enzyme substrate; gene deletion mutation; gene mutation; genetic recombination; human genetic material tag; laboratory mouse; laboratory rabbit; neoplastic cell; nonhistone nucleoprotein; nuclear magnetic resonance spectroscopy; nucleic acid sequence; phosphorylation; protein kinase; protein structure function; site directed mutagenesis; synthetic peptide; tissue /cell culture; transfection

The long-term objective of the research is to determine the structural basis underlying the suggested biological effects mediated by mammalian HMG-I proteins. HMG-I is the principal member of an isoform group (the HMGI family) of the nuclear proteins and is the first mammalian nonhistone protein demonstrated to specifically bind, both in vitro and in vivo, to A T-rich regions of DNA. Recent reports indicate that all types of cancerous cells contain exceptionally high concentrations of HMGI proteins which have been proposed to be biochemical markers for the transformed state. In vitro, members of the HMGI family have been demonstrated to act as gene transcription stimulatory factors and also to specifically bind to important A.T-rich DNA regulatory regions, including gene promoters, enhancers and the DNA replication origins of several organisms. A peptide "binding domain" (BD) common to all known HMGI proteins has been identified that mediates binding of these proteins to the narrow minor groove of DNA and is called "A T-hook" because of its novel predicted secondary structure. In certain ways the BD peptide resembles the antitumor/antiviral drugs distamycin, netropsin and the dye Hoechst 33258, ligands which effectively compete with HMGI proteins for DNA binding both in vitro and in vivo. Individual HMGI proteins have three separate BD peptide motifs. Both in vitro and in vivo, HMGI proteins are preferred substrates for the cell division regulating enzyme cdc2 kinase which specifically phosphorylates the "hook" region of the BD peptides. In vitro such phosphorylation has been demonstrated to significantly weaken the DNA binding affinity of both synthetic BD peptides and intact HMGI proteins. Specific Aims of Project: (1) To determine the 3D solution structure of both the unphosphorylated and cdc2 kinase phosphorylated synthetic BD peptides employing 2D NMR techniques; (2) Determine the effects of specific deletions, duplications or mutations in one or more of the individual BD peptides present in wild type (wt) HMG-I proteins on the ability of such mutant proteins to strongly and specifically interact with A T-rich DNA. (3) Produce recombinant hybrid BD-peptide/"tag-peptide" fusion proteins and determine whether these hybrid proteins can specifically "target" and bind to stretches of A T-rich sequence in vitro and in vivo. The results of these studies will not only help establish the structural basis for understanding the biological effects of an important group of proteins, but will also contribute new detailed information about general molecular mechanisms involved in specific DNA-protein interactions. And, perhaps as importantly, the experiments may establish a mechanism for "targeting" specific peptides or proteins A T-rich sequences in living cells.

该研究的长期目的是确定结构 哺乳动物介导的建议的生物学作用的基础 HMG-I蛋白质。 HMG-I是同工型组的主要成员（ 核蛋白的HMGI家族），是第一个哺乳动物非组蛋白 蛋白质证明可以在体外和体内特异性结合 DNA的t富区域。 最近的报告表明所有类型的癌性 细胞包含具有极高浓度的HMGI蛋白 被认为是转化状态的生化标记。 在 体外，HMGI家族的成员已被证明充当基因 转录刺激因子，并专门结合 重要的A.T富含DNA调节区，包括基因启动子， 增强剂和几种生物的DNA复制起源。 肽 已经确定了所有已知HMGI蛋白共有的“结合域”（BD） 介导这些蛋白质与DNA的狭窄小凹槽的结合 由于它的小说预测次要，因此被称为“ t钩” 结构。 在某些方面，BD肽类似于 抗肿瘤/抗病毒药物大霉素，Netropsin和Dye Hoechst 33258， 有效与HMGI蛋白竞争DNA结合的配体 体外和体内。 单个HMGI蛋白具有三个独立的BD 肽图案。 在体外和体内，HMGI蛋白都是首选 调节酶CDC2激酶的细胞分裂底物 特异性地磷酸化BD肽的“钩子”区域。 体外 这种磷酸化已被证明可以显着削弱DNA 合成BD肽和完整HMGI蛋白的结合亲和力。 项目的具体目的：（1）确定3D解决方案结构 未磷酸化和CDC2激酶磷酸化的合成BD 采用2D NMR技术的肽； （2）确定特定的影响 一个或多个单个BD中的删除，重复或突变 野生型（WT）HMG-I蛋白的肽对这种能力 突变蛋白与富含T的DNA强，并特别相互作用。 （3）产生重组杂交BD肽/“ Tag肽”融合蛋白和 确定这些混合蛋白是否可以特异性“靶”并结合 在体外和体内伸展富含T的序列。 结果 这些研究不仅将有助于建立结构性基础 了解一组重要蛋白质的生物学作用，但 还将贡献有关通用分子的新详细信息 参与特定DNA蛋白相互作用的机制。 而且，也许是 重要的是，实验可以建立“靶向”的机制 特定的肽或蛋白质在活细胞中富含T的序列。