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: 3305772
• 负责人: RAYMOND REEVES
• 金额: $ 14.47万
• 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/3305772
• 关键词: DNA binding protein; DNA footprinting; cell cycle; cell cycle proteins; chimeric proteins; enzyme substrate; gene deletion mutation; gene mutation; genetic recombination; human genetic material tag; laboratory mouse; laboratory rabbit; neoplastic cell; 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.

研究的长期目标是确定结构 所提出的哺乳动物介导的生物效应的基础 HMG-I蛋白。 HMG-I是同种型组（HMG-I的主要成员）的主要成员。 HMGI家族）的核蛋白，是第一个哺乳动物非组蛋白 在体外和体内，蛋白质被证明特异性结合A 富含T的DNA区域。 最近的报告表明，所有类型的癌症 细胞含有异常高浓度的HMGI蛋白， 被认为是转化状态的生化标志物。 在 在体外，HMGI家族的成员已被证明作为基因 转录刺激因子，并特异性结合 重要的富含A.T.的DNA调控区，包括基因启动子， 增强子和几种生物体的DNA复制起点。 的肽 已经鉴定了所有已知HMGI蛋白共有的“结合结构域”（BD 介导这些蛋白质与DNA的狭窄小沟的结合 它被称为“T型钩”，因为它的新的预测次级 结构 在某些方面，BD肽类似于 抗肿瘤/抗病毒药物偏端霉素、netropsin和染料Hoechst 33258， 与HMGI蛋白有效竞争DNA结合的配体， 在体外和体内。 单个HMGI蛋白具有三个独立的BD 肽基序。 在体外和体内，优选HMGI蛋白 细胞分裂调节酶CDC 2激酶的底物 特异性磷酸化BD肽的“钩”区。 体外 这种磷酸化作用已被证明能显著削弱DNA 合成BD肽和完整HMGI蛋白的结合亲和力。 项目具体目标：（1）确定三维解决方案结构， 未磷酸化和cdc 2激酶磷酸化的合成BD 肽采用二维核磁共振技术;（2）确定特定的影响， 在一个或多个个体BD中的缺失、重复或突变， 存在于野生型（wt）HMG-I蛋白中的肽对这种 突变蛋白与富含T的DNA强烈且特异性地相互作用。 (3)产生重组杂合BD-肽/“标签-肽”融合蛋白， 确定这些杂交蛋白是否可以特异性地“靶向”并结合 在体外和体内对富含T的序列的延伸。 的结果 这些研究不仅有助于建立结构基础， 了解一组重要蛋白质的生物学效应，但 还将提供关于一般分子的新的详细信息， 参与特定的DNA-蛋白质相互作用的机制。 也许， 重要的是，这些实验可能建立一种“靶向”机制， 活细胞中富含T的序列。