S POMBE PROTEIN/CENTROMERIC DNA INTERACTION

描述（改编自申请人摘要）：详细研究定义 复制、修复和重组的复杂性已经阐明 参与这些复杂的蛋白质的作用 流程. 许多细节仍有待解决。 该地区只有 最近开始在生物化学水平上受到攻击，涉及有丝分裂， 重要的生物学过程，染色体被分类，配对， 精确地分配到每个子细胞。 在有丝分裂期间， 指导染色体的运动。 着丝粒是细胞分裂的位点， 动粒形成，它是姐妹染色单体保留的最后一个位置 在中期-后期过渡期分离之前结合。 大量证据表明，有丝分裂检查点 控制在调节细胞周期中起重要作用。 各种 干扰有丝分裂纺锤体组装的药物阻断了细胞周期， 分裂。 有丝分裂检查点目标已经成为许多研究的焦点。 抗肿瘤药物 因此，深入了解其背后的机制 有丝分裂过程可能导致更好地理解异常 增长 该提案旨在确定与蛋白质相互作用的蛋白质。 分离自粟酒裂殖酵母的着丝粒DNA。 S.粟酒含有 3条染色体和3个不同的着丝粒。 着丝粒的序列 DNA已在很大程度上被确定，它们的大小从约40到约100个不等。 大于100 kb。 S.粟酒酵母没有那么复杂， 高等真核生物中的那些（300-5000 kbp），但比 S.酿酒酵母染色体作为着丝粒。 而与S.酿酒酵母 和哺乳动物已经被定义，几乎没有蛋白质相互作用， 的着丝粒。有报道称， 调查人员发现 结合双链体DNA中AT富集区的许多蛋白质。 这些 蛋白质基于它们与衍生的起始序列的结合而被分离 从S.粟球，克隆后 转化为质粒，支持质粒复制。 其中三种蛋白质 克隆和序列测定 其中两种蛋白质称为ARS结合蛋白 蛋白1（abp 1）和着丝粒结合蛋白B同源物（Cbh）， 它们彼此高度同源，并且都具有与 哺乳动物着丝粒DNA结合蛋白CENP-B Abp 2蛋白 与任何已知蛋白质都没有同源性，但含有一个基序特征， 富含AT的DNA结合蛋白。 蛋白质Cbh 1已被证明与 S.粟酒裂殖子着丝粒DNA，并似乎发挥功能作用， 稳定S.粟酒 调查人员计划进一步 表征这些蛋白质在结合S. 粟酒属着丝粒DNA序列，确定其核定位， 利用它们作为鉴定其他着丝粒蛋白的手段， 它们在着丝粒DNA交易和功能中的作用。

DESCRIPTION (Adapted from applicant's abstract): Detailed studies defining the complexities of replication, repair and recombination have elucidated the role of individual proteins that participate in these complicated processes. Many of the details remain to be solved. The area that has only recently begun to be attacked at the biochemical level concerns mitosis, the important biological process by which chromosomes are sorted, paired and accurately distributed to each daughter cell. During mitosis centromeres direct the movement of chromosomes. The centromere is the site at which the kinetochore forms and it is the last site by which sister chromatids remain associated prior to their separation at the metaphase-anaphase transition. Considerable evidence has accumulated indicating that mitotic checkpoint controls play an important role in regulating the cell cycle. A variety of drugs that interfere with mitotic spindle assembly block the cell cycle in mitosis. Mitotic checkpoint targets have been the focus of a number of antitumor drugs. Thus, a thorough understanding of the mechanism underlying the process of mitosis may lead to a better understanding of aberrant growth. This proposal is aimed at identifying proteins that interact with centromeric DNA isolated from Schizosaccharomyces pombe. S. pombe contains 3 chromosomes and 3 distinct centromeres. The sequence of the centromeric DNAs have been largely determined and they vary in size from about 40 to greater than 100 kb. The centromeres of S. pombe are less complex than those in higher eukaryotes (300-5000 kbp) but considerably more complex than the 125 bp region on S. cerevisiae chromosomes that functions as centromere. While a number of proteins that interact with centromeres of S. cerevisiae and mammals have been defined, virtually no proteins interacting with centromeres of S. pombe have been reported. The investigators have detected a number of proteins that bind to AT-rich regions in duplex DNA. These proteins were isolated based on their binding to origin sequences derived from autonomous recognition sequences (ARSs) of S. pombe, which when cloned into plasmids, support plasmid replication. Three of the proteins have been cloned and sequenced. Two of the proteins, which are called ARS binding protein 1 (abp1) and centromere binding protein B homologue (Cbh), are highly homologous to one another and both possess significant homology to the mammalian centromere DNA binding protein, CENP-B. The protein Abp2 shares no homology to any known protein but contains a motif characteristic of AT-rich DNA binding proteins. The protein Cbh1 has been shown to bind to S. pombe centromeric DNA and appears to play a functional role in stabilizing chromosomes in S. pombe. The investigators plan to further characterize these proteins regarding their specificity in binding to S. pombe centromeric DNA sequences, determine their nuclear localization, utilize them as means to identify other centromere proteins and examine their role in centromeric DNA transactions and functions.