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

研究的长期目标是确定结构 哺乳动物介导的生物效应的基础 HMG-I蛋白。HMG-I是异构体基团( HMGI家族)，是第一个哺乳动物非组蛋白 体外和体内证明能与A特异性结合的蛋白质 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，这些蛋白具有 被认为是这种转化状态的生化标记。在……里面 在体外，HMGI家族的成员已经被证明是基因 转录刺激因子也能与之特异性结合 重要的富含A.T的DNA调节区，包括基因启动子， 几种生物的增强剂和DNA复制起源。A肽 已确定所有已知HMGI蛋白共有的“结合结构域”(BD) 它介导这些蛋白质与DNA的狭窄小沟结合 因其新奇的预测次要因素而被称为“T形钩” 结构。在某些方面，BD多肽类似于 抗肿瘤/抗病毒药物地塞霉素、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蛋白有三个独立的BD 多肽图案。无论在体外还是在体内，HMGI蛋白都是首选的 细胞分裂调节酶cdc2激酶的底物 特别是使BD多肽的“挂钩”区域磷酸化。离体 这种磷酸化已被证明显著削弱了DNA 合成的BD多肽和完整的HMGI蛋白的结合亲和力。 项目的具体目标：(1)确定项目的三维解结构 非磷酸化和cdc2激酶均可磷酸化合成BD 多肽的2D核磁共振技术；(2)确定特异性的影响 一个或多个个体BD的缺失、复制或突变 野生型(Wt)HMG-I蛋白中存在的多肽对这种能力的影响 突变的蛋白质与富含T的DNA强烈和特异地相互作用。 (3)制备重组杂合BD-肽/“标签-肽”融合蛋白 确定这些杂合蛋白是否能特异性地“靶向”并结合 A富含T的序列在体外和体内的延伸。结果是 这些研究不仅将有助于为 了解一组重要蛋白质的生物效应，但 还将提供有关一般分子的新的详细信息 涉及特定DNA-蛋白质相互作用的机制。而且，也许就像 重要的是，这些实验可能会建立一种“定向”机制。 特定的多肽或蛋白质：活细胞中富含T的序列。

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.