DNA POLYMERASES

由于DNA序列的变化具有深远的遗传后果，因此 了解维护和复制的机制很重要 如果我们要完全了解遗传病的基础，这些序列 可能还有退化性疾病，如癌症和衰老。DNA 聚合酶显然是DNA新陈代谢的关键角色，这也是我们的目标 为了了解这些酶如何有助于维持遗传 正直。 几年前，我们鉴定并提纯了一种DNA聚合酶，Polepsilon， 在渗透性二倍体人类中介导DNA修复合成 成纤维细胞。然而，Akio和他的同事们现在建议，在 酵母、Polepsilon参与DNA复制。这项提议是 继续从HeLa文库中克隆并测序 与酵母polepsilon基因同源。将使用序列信息 以制造针对cDNA蛋白的抗体，并将利用抗体来 确认此cDNAs为polepsilon的cdna，并对该酶进行纯化。 确定与该酶结合的其他蛋白质。信使核糖核酸、蛋白质和活性 将通过细胞周期监测Polepsilon的变化，并监测 在非周期HeLa细胞和分化的神经母细胞瘤细胞中 就像暴露在DNA损伤剂中的细胞一样。最后，催化性能 和一种具有较小催化亚基的酶(S) 将与它们的亚基结构进行比较。最终，我们希望 理解(S)Polepsilon在DNA修复和延伸中可能起到的作用 在DNA复制过程中。 同时，对PERSα、β、Delta和 将对Epsilon进行有条不紊的研究，并移除RNA引物和 用多聚贝塔、DNA酶V和核糖核酸酶H的复合体取代DNA将 在模型系统中进行研究。我们之前对HeLa DNA的经验 聚合酶α、β和epsilon，并作为一种 副产品使我们处于一个独特的境地，每种产品都有库存 使这些酶与底物的催化性能进行比较 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新陈代谢中。

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.