MAMMALIAN-LIKE REPLICATION ORIGIN IN S POMBE

真核细胞在细胞周期的S期复制它们的DNA分子。 细胞周期 双向复制在称为 “起源” 虽然有些起源于S期早期， 在S阶段之后，每个原点在单个S阶段内仅发射一次。 因为正常的细胞分裂需要受调控的DNA复制， 因为不正确的复制调控导致了基因组 涉及恶性进展的不稳定性，改善理解 DNA复制的调节可能有助于我们 了解癌症和其他涉及细胞的健康状况 师. 了解真核生物DNA复制的机制 受监管的将需要说明的DNA序列， 原点函数 先前的研究表明， 芽殖酵母，酿酒酵母，包括，最低限度地，短的 序列（ARS共有序列，ACS）和更长的侧翼序列 其容易解绕，但在其它方面从原点到原点是可变的 (the DNA解旋元件，DUE）。 这种序列元素的组合 对于质粒中的起始活性是足够的。 序列允许 质粒中的起始活性被称为ARS元件。 在其他真核生物中， 需要原始函数。 这种信息的缺乏部分是由于 事实上，在其他测试的真核生物（哺乳动物细胞）中， 和果蝇），常用的起源映射技术，两个 二维（2D）凝胶电泳，提供的结果表明， 复制在整个区域启动，而不是在一个精确的 绝佳的价钱 此外，很难测试假定的功能起源， 在果蝇和哺乳动物细胞中， 自主的细胞质复制（ARS功能）或容易引入 通过同源重组将序列插入基因组。 我们刚刚发现了一个复制起点（ura 4起点）， 裂殖酵母，粟酒裂殖酵母，我们已经证明， 类似于哺乳动物细胞和果蝇的起源， 凝胶电泳表明起始发生在一个宽的区域。 然而，与果蝇和哺乳动物细胞不同，S. pombe支持两者 ARS功能和同源重组进入基因组。 在本申请中，我们建议利用这些实验优势 为了确定ura 4起源顺式所需的最小DNA片段， 函数（最小原点），以确定重要的连续性 在最小的起源，使用体内和染色质足迹 技术来确定蛋白质-DNA相互作用的最小起源 区域，并纯化和表征蛋白质结合到最小的 起源 由于ura 4起源和 哺乳动物/果蝇的起源，我们怀疑所获得的数据将 证明有助于了解真核生物的复制 起源一般。

Eukaryotic cells duplicate their DNA molecules during the S phase of the cell cycle. Bidirectional replication initiates at sites called "origins". Although some origins fire in early S phase and others in later S phase, each origin fires only once within a single S phase. Because regulated DNA replication is required for proper cell division, and because incorrectly regulated replication contributes to the genomic instability implicated in malignant progression, improved comprehension of regulation of DNA replication is likely to contribute to our understanding of cancer and other health conditions which involve cell division. Understanding the mechanisms by which eukaryotic DNA replication is regulated will require elucidation of the DNA sequences which specify origin function. Previous studies have revealed that origins in the budding yeast, Saccharomyces cerevisiae, consist, minimally, of a short sequence (the ARS consensus sequence, ACS) and a longer flanking sequence which is easily unwound but is otherwise variable from origin to origin (the DNA Unwinding Element, DUE). This combination of sequence elements is sufficient for origin activity in plasmids. Sequences permitting origin activity in plasmids are called ARS elements. In other eukaryotic organisms, nothing is known about the sequences required for origin function. This dearth of information is partly due to the fact that, in other tested eukaryotic organisms (mammalian cells and Drosophila), the commonly used origin mapping technique, two dimensional (2D) gel electrophoresis, provides results which suggest that replication initiates throughout a broad zone rather than at a precise site. In addition, it is difficult to test putative origins for function in Drosophila and mammalian cells; neither organism reproducibly supports autonomous plasmic replication (ARS function) or facile introduction of sequences into the genome by homologous recombination. We have just discovered a replication origin (the ura4 origin) in the fission yeast, Schizosaccharomyces pombe, and we have shown that it resembles origins in mammalian cells and Drosophila in the sense that 2D gel electrophoresis suggests that initiation occurs in a broad zone. However, unlike Drosophila and mammalian cells, S. pombe supports both ARS function and homologous recombination into the genome. In this application, we propose to utilize these experimental advantages to identify the minimal stretch of DNA required in cis for ura4 origin function (the minimal origin), to identify the important subsequences within the minimal origin, to use in vivo and chromatin footprinting techniques to identify protein-DNA interactions in the minimal origin region, and to purify and characterize proteins which bind to the minimal origin. Because of the resemblance between the ura4 origin and mammalian/Drosophila origins, we suspect that the data obtained will prove useful in developing an understanding of eukaryotic replication origins in general.