S POMBE PROTEIN/CENTROMERIC DNA INTERACTION

描述(改编自申请人的摘要)：详细研究定义 复制、修复和重组的复杂性已经阐明 参与这些复杂过程的单个蛋白质的作用 流程。许多细节仍有待解决。仅有的区域 最近开始在生化水平上受到攻击，涉及有丝分裂， 重要的生物学过程，染色体通过它来分类、配对和 准确地分配到每个子细胞。在有丝分裂着丝粒期间 指导染色体的运动。着丝粒是指 动粒形成，是姐妹染色单体保留的最后一个部位 在中期-后期转变时，在分离之前是伴生的。 大量证据表明，有丝分裂检查点 调控在调节细胞周期方面发挥着重要作用。各种各样的 干扰有丝分裂纺锤体组装的药物阻止细胞周期 有丝分裂。有丝分裂检查点目标一直是许多 抗肿瘤药物。因此，对潜在的机制有一个彻底的了解 有丝分裂的过程可能导致对异常的更好的理解 成长。这项提议的目的是识别与之相互作用的蛋白质 从裂殖酵母中分离到着丝粒DNA。金黄色葡萄球菌含有 3条染色体和3条不同的着丝粒。着丝粒的序列 DNA在很大程度上已经确定，它们的大小从大约40到 大于100 kb。庞氏链霉菌的着丝粒比 在高等真核生物中(300-5000 kbp)，但比 酿酒酵母染色体上作为着丝粒的125个碱基对的区域。 而一些与酿酒酵母着丝粒相互作用的蛋白质 哺乳动物已经被定义为，几乎没有与之相互作用的蛋白质 庞氏链霉菌的着丝粒已有报道。调查人员发现 与双链DNA中富含AT的区域结合的一些蛋白质。这些 根据它们与起始序列的结合来分离蛋白质 来自S.pombe的自主识别序列(ARSS)，当克隆时 转化为质粒，支持质粒复制。其中三种蛋白质已经被 克隆并测序。其中两种蛋白质称为ARS结合 蛋白1(Abp1)和着丝粒结合蛋白B同源物(CBH)，是 彼此高度同源，并且两者都具有显著的同源性 哺乳动物着丝粒DNA结合蛋白CENP-B。Abp2蛋白 与任何已知蛋白质没有同源性，但包含基序特征 富含AT的DNA结合蛋白。Cbh1蛋白已被证明与 该基因在Pombe着丝粒DNA中起重要作用 稳定庞氏链霉菌的染色体。调查人员计划进一步 鉴定这些蛋白与S。 Pombe着丝粒DNA序列，确定它们的核定位， 利用它们作为手段来鉴定其他着丝粒蛋白并检测 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.