ENZYMATIC MECHANISMS OF DNA REPLICATION

将采用一种组合遗传生化方法来研究dna。 在酵母中复制。这两个学科的发展方向 合并后的基因将被称为“反向遗传学”。被驱逐的人预计会 参与酵母DNA复制的酵母DNA将通过特定的检测得到纯化 根据以前的知识，抗体或寡核苷酸将是 制备并克隆了相应的基因。基因置换与基因 突变技术将被用来构建可用于 确定单个分离物是否真的参与酵母 复制与否。这些被驱逐的人将被用来重建 在含有ARSL序列的质粒DNA上复制，推测是一个 染色体复制子。在这项工作中，我们克隆了基因并制作了 复制DNA聚合酶催化亚基DNA中的突变体 聚合酶I将被广泛应用于基因和过量生产 蛋白质在全酶中的特性。我们还分离出了一种蛋白质 它与含有ARS1共识序列的DNA特异结合。 这种蛋白质是一种特定的启动蛋白的很好的候选者，而这 将通过将其提纯到均一，制备抗体来进行研究 并克隆该基因。聚合酶全酶和特异性启动子 来自任何重建系统关键部分的蛋白质。两种缺失的蛋白质 真核复制中的环节是解旋酶和复制体 单链DNA结合蛋白。隔离这些问题的方法将是 描述并一旦获得它们，将应用反向遗传学。 已经提纯了几个SSB，克隆了它们的基因并进行了研究 变种人准备好了。其他蛋白质或复制将通过以下方式纯化 其他人发表的用作试剂的程序：RNaseH，Primase， 拓扑异构酶等。由于底物酶基因将非常有用，它 将从表达库中克隆。最终，我们希望了解 DNA复制子的使用是如何受到监管的，我们将通过询问如何做到这一点 这些蛋白质和基因受到调控，并通过进一步研究 DNA序列，ARSL。

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.