ENZYMATIC MECHANISMS OF DNA REPLICATION

DNA的研究将采用遗传生化结合的方法 酵母中的复制 这两个学科将如何 将被称为“反向遗传学”。“搬运工们希望 参与酵母DNA复制的细胞将通过特定的测定进行纯化 基于先前的知识，抗体或寡核苷酸将 制备并克隆相应的基因。 基因置换与基因 干扰技术将用于构建突变体， 确定单个分离株是否真的参与了酵母菌 复制与否。 那些涉及的门户将被用来重建 在含有ARS1序列的质粒DNA上的复制，推测是一种 染色体复制因子 为了这项工作，我们克隆了基因， 复制型DNA聚合酶催化亚单位的突变体 聚合酶I 将广泛利用基因和过度生产的 蛋白质在表征全酶。 我们还分离出一种蛋白质 与含有ARS1共有序列的DNA特异性结合。 该蛋白是特异性起始蛋白的良好候选物， 将通过将其纯化至均一，制备抗体 并克隆该基因。 聚合酶全酶和特异性引发剂 蛋白质从任何重组系统的关键。 两种缺失的蛋白质 真核生物复制中的环节是解旋酶和复制酶。 单链DNA结合蛋白。 分离这些的方法将是 一旦获得，将应用反向遗传学。 一些SSBs已经被纯化，它们的基因被克隆和研究 变种人准备好了 其他蛋白质或复制将被纯化， 其他人公开的用作试剂的方法：RNase H，引物酶， 由于引物酶的基因将是非常有用的， 将从表达文库中克隆。 最终我们希望了解 DNA复制子的使用是如何被调节的，我们将通过询问如何来做到这一点。 这些蛋白质和基因受到调控， DNA序列，ARS1。

A combined genetic biochemical approach will be taken to the study of DNA replication in yeast. The way in which these two disciplines will be combined will be called "reverse genetics." The porteins expected to participate in yeast DNA replication will be purified by specific assays based on previous knowledge, antibodies or oligonucleotides will be prepared and the corresponding genes cloned. Gene replacement and gene distuption techniques will be used to construct mutants useful in establishing whether the individual isolates actually participate in yeast replication or not. Those porteines involved will be used to reconstitutue replication on plasmid DNAS CONTAINING THE ARSl sequence, presumably a chromosomal replicator. For this work, we have cloned the gene and made mutants in the catalytic subunit of the replicative DNA polymerase, DNA polymerase I. Extensive use will be made of the gene and the overproduced protein in characterizing the holoenzyme. We have also isolated a protein that binds specifically to DNAs containing the ARS1 consensus sequence. This protein is a good candidate for a specific initiation protein and this will be investigated by purifying it to homogeneity, preparing antibodies and cloning the gene. The polymerase holoenzyme and the specific initiator protein from the crux of any reconstitution system. Two missing protein links in eukaryotic replication are the helicase and a replicative single-stranded DNA binding protein. Ways to isolate these will be described and once they are obtained reverse genetics will be applied. Several SSBs have already been purified, their genes cloned and intresting mutants prepared. Other proteins or replication will be purified by procedures published by others to be used as reagents: RNase H, primase, topoisomerases etc. Since the gene for primase would be very useful, it will be cloned from a expression library. Ultimately we wish to understand how DNA replicon useage is regulated and we will do this by asking how these proteins and genes are regulated as well as by further studying the DNA sequence, ARSl.