DNA POLYMERASES

由于DNA序列的变化具有深刻的遗传后果， 重要的是要了解维护和复制的机制， 如果我们想完全了解遗传疾病的基础 以及可能的退行性疾病如癌症和衰老。 的DNA 聚合酶显然是DNA代谢中的关键角色，这也是我们的目标。 为了了解这些酶是如何帮助维持遗传的， 完整 几年前，我们鉴定并纯化了一种DNA聚合酶， 在透化的二倍体人类中， 成纤维细胞 然而，Akio Sugino和同事现在建议， 酵母中，聚合酶参与DNA复制。 有关建议是 继续从HeLa文库中克隆和测序cDNA， 与酵母聚合酶基因同源。 将使用序列信息 以制备cDNA蛋白的抗体，抗体将用于 确认cDNA是聚合酶的cDNA，纯化酶， 识别与酶结合的其他蛋白质。 mRNA、蛋白质和活性 将通过细胞周期监测聚合物的浓度， 在非周期性HeLa细胞和分化的神经母细胞瘤细胞中， 就像暴露在DNA破坏剂中的细胞一样。 最后，催化性能 和具有较小催化亚基的酶的形式 将与它们的亚基结构进行比较。 最终，我们希望 了解聚合酶在DNA修复和延伸中的作用 在DNA复制过程中。 同时，通过pols α、β、δ和 将系统地研究DNA，并去除RNA引物， 用polbeta、DNase V和RNase H的复合物替换DNA， 在模型系统中进行研究。 我们以前使用HeLa DNA的经验 聚合酶α、β和pol δ的积累， 副产品使我们处于一种独特的境地， 这些酶和底物， 可以在单个实验中进行。 通过这种方式，我们希望 有助于了解单个DNA聚合酶的作用 在DNA代谢中。

Since changes of DNA sequence have profound genetic consequences, it is important to understand the mechanisms for maintaining and replicating these sequences if we are to understand fully the basis of genetic diseases and possibly degenerative diseases such as cancer and aging. The DNA polymerases are obviously key players in DNA metabolism, and it is our goal to understand how these enzymes contribute to maintaining genetic integrity. Several years ago we identified and purified a DNA polymerase, pol epsilon, that served to mediate DNA repair synthesis in permeabilized diploid human fibroblasts. However, Akio Sugino and coworkers have now suggested that in yeast, pol epsilon participates in DNA replication. the proposal is to continue to clone and to sequence a cDNA from a HeLa library with strong homology to the yeast pol epsilon gene. Sequence information will be used to make antibodies to the cDNA protein and antibodies will be utilized to confirm that the cDNA is that of pol epsilon, to purify the enzyme, and to identify other proteins bound to the enzyme. mRNA, protein, and activity of pol epsilon will be monitored through the cell cycle and also monitored in non-cycling HeLa cells and in differentiated neuroblastoma cells as well as in cells exposed to DNA damaging agents. Finally, catalytic properties of pol epsilon and a form(s) of the enzyme with a smaller catalytic subunit will be compared as will their subunit structures. Ultimately we wish to understand what role(s) pol epsilon might have in DNA repair and elongation during DNA replication. Meanwhile, the utilization of RNA primers by pols alpha, beta, delta and epsilon will be methodically studied and removal of RNA primers and replacement with DNA by a complex of polbeta, DNase V, and an RNase H will be studied in model systems. Our previous experience with HeLa DNA polymerases alpha, beta and epsilon, and an accumulation of pol delta as a byproduct has placed us in a unique situation of having stocks of each of these enzymes and substrates so that comparisons of catalytic properties can be carried out in single experiments. In this manner, we hope to contribute to the knowledge of the roles of the individual DNA polymerases in DNA metabolism.