THREE-DIMENSIONAL STRUCTURE OF THE ENDS OF CHROMOSOMES

染色体末端的三维结构

• 批准号: 3123538
• 负责人: STEVE C SCHULTZ
• 金额: $ 18.23万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-30 至 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3123538
• 关键词: DNA binding protein; X ray crystallography; cell cycle; cell senescence; crystallization; gene mutation; genetic regulation; nucleic acid sequence; nucleoproteins; protein structure function; protozoa; telomere; yeasts

The overall goal of this project is to learn about telomere structure and function by determining the x-ray crystal structures of nucleoprotein complexes that compose telomeres. Currently, our efforts ar focused on two cloned and characterized proteins that are known to be components of telomeres in vivo: (i) the telomere binding protein from Oxytricha nova which interacts with single-strand DNA at the very ends of telomeres and (ii) RAP1 from yeast which interacts with the double-stranded DNA region of telomeres and has been shown to participate in regulating telomere length. The telomere binding protein from Oxytricha nova interacts with single- strand DNA (sequence TTTTGGGGTTTTGGGG) that occurs at the very ends of telomeres. The telomere binding protein consists of two subunits; a 56 Kd alpha-subunit that interacts single-strand (TTTTGGGG)n DNA in a sequence specific manner and a 41 KD beta-subunit that alters the DNA in a sequence specific manner and a 41 KD beta-subunit that alters the DNA binding properties of the alpha-subunit. The alpha- and beta-subunits do not interact in solution in the absence of DNA, but together with (TTTTGGGG)n DNA they form a very tight ternary complex containing the alpha-subunit, the beta-subunit, and DNA. We have obtained crystals of (i) the alpha-subunit complexed with TTTTGGGG DNA that diffract to 2.7A resolution, (ii) the beta-subunit in th presence of GGGG DNA, and (iii) the entire ternary complex containing the alpha-subunit, the beta- subunit, and GGGGTTTTGGGG DNA that diffract to at least 3 A resolution. We are now poised for a very detailed look at the telomere binding protein-DNA complex which will greatly enhance our understanding of how the two subunits of this protein interact with DNA and with each other to form the nucleoprotein complex that caps the ends of chromosomes. RAP1 is associated with yeast telomeres in vivo and is necessary for proper regulation of telomere length. Mutations in this protein give rise to either elongated or shortened telomeres. RAP1 is a sequence- specific double-stranded DNA binding protein that interacts with the sequence ACACCCACACACC which occurs every 30-40 base pairs in yeast telomeres. Interestingly, RAP1 has been shown to bend DNA such that it may give rise to a higher order structure in yeast telomeres. We have begun efforts to obtain large quantities of RAP1 for crystallization and structure determination. That improper maintenance of telomere length and structure limits a cells ability to divide and results in senescence has been clearly established. Indeed, it is proposed that telomeres play a critical role in cell division and in the "senescence program:\" in higher eukaryotes. Understanding the structure of the nucleoprotein complexes that compose telomeres will lead to a better overall understanding of the structure and function of telomeres.

该项目的总体目标是了解端粒结构， 通过确定核蛋白的X射线晶体结构 组成端粒的复合体。 目前，我们的工作重点是 两个克隆和表征的蛋白质，已知是 体内端粒：（i）来自Oxytricha nova的端粒结合蛋白 它与端粒末端的单链DNA相互作用， (ii)与双链DNA区域相互作用的来自酵母的RAP 1 并已被证明参与调节端粒 长度 来自Oxytricha nova的端粒结合蛋白与单个- 链DNA（序列TTTTGGGGTTTTGGGG），发生在 端粒 端粒结合蛋白由两个亚基组成： 与单链（TTTTGGGG）n DNA相互作用的Kd α-亚基， 序列特异性的方式和一个41 KD的β亚基，改变DNA， 一种序列特异性方式和一个41 KD的β亚基， α-亚基的结合特性。 α和β亚基 在没有DNA的情况下，在溶液中不相互作用，但与 （TTTTGGGG）n DNA，它们形成非常紧密的三元复合物， α亚基β亚基和DNA 我们已经获得了 (i)与TTTTGGGG DNA复合α-亚基与2.7A 分辨率，（ii）在GGGG DNA存在下的β亚基，和（iii） 整个三元复合物包含α-亚基，β- 亚基和GGGGTTTTGGGG DNA。 我们现在准备对端粒结合进行非常详细的研究 蛋白质-DNA复合物，这将大大提高我们的理解， 这种蛋白质的两个亚基与DNA相互作用， 形成覆盖染色体末端的核蛋白复合体。 RAP 1在体内与酵母端粒相关，并且是 端粒长度的适当调节。 这种蛋白质的突变 端粒变长或变短。 RAP 1是一个序列- 特异性双链DNA结合蛋白，与 在酵母中每30-40个碱基对出现一次的序列ACACCCACACACC 端粒 有趣的是，RAP 1已被证明可以弯曲DNA， 可能导致酵母端粒中的高级结构。 我们有 开始努力获得大量RAP 1用于结晶， 结构测定 端粒长度和结构的不适当维护限制了a细胞 已经清楚地建立了分裂和导致衰老的能力。 事实上，有人提出端粒在细胞中起着关键作用， 在高等真核生物中的衰老程序中。 了解组成核蛋白复合物的结构 端粒将有助于我们更好地了解 和端粒的功能。