DNA POLYMERASES

描述：由于DNA序列的变化具有深刻的遗传学意义， 重要的是要了解维持的机制。 并复制这些序列，如果我们要完全理解 遗传和变性疾病及其治疗的可能目标。 DNA聚合酶显然是DNA代谢的关键参与者， 申请人的目标是了解这些酶如何有助于 保持基因的完整性 几年前，这个研究小组鉴定并纯化了一种DNA聚合酶（polymerase，polymerase）。 在透化细胞中， 二倍体人成纤维细胞。 然而，现在有人提出，在 在酵母中，聚合酶参与DNA复制和DNA修复。 然而，虽然来自酵母和HeLa的“核心”聚合物具有类似的 催化，260 kDa的亚基，它们有不相关的辅助亚基。 到 了解哺乳动物细胞中的聚合酶的作用，这个实验室非常 最近生产了一组抗HeLa细胞的单克隆抗体。 在 这样做，针对p260的单克隆抗体的子集，另一个“核心”， 亚基，p55，以及显示复合聚合酶肽 (p85，p70，p49）。 p85和p70被鉴定为Ku 自身抗原，一种涉及重组和重组修复的蛋白质。 利用这些抗体，p55将被测序、克隆和过表达。 与P260。 将鉴定和克隆p49。 的意义 将研究与Ku蛋白复合的聚合酶， p350蛋白激酶（其形成Ku相关DNA依赖性蛋白 激酶）也将确定与聚合酶的复合物。 从这些 结果希望了解是否以及如何参与pollution 在对DNA损伤的应激反应中（例如，它会不会在 复制并要求响应，或者它可能被招募来修复， 这种试剂RNA聚合II？）。 单克隆抗体也将被用于合作，以辨别作用 人聚合酶的核苷酸切除修复，重组修复， 不匹配修复、复制和检查点控制。 复制旁路 分析将确定聚合酶在损伤旁路中的可能作用 复制的 最后，一种新的线粒体外HeLa DNA聚合酶， 类似于pols β和γ，将用特殊的 注意它是否是酵母pol β的同源物或核形式 关于PolGamma

DESCRIPTION: 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 and degenerative diseases and possible targets for their treatment. The DNA polymerases are obviously key players in DNA metabolism and it is the applicant's goal to understand how these enzymes contribute to maintaining genetic integrity. Several years ago this group identified and purified a DNA polymerase (pol epsilon) that served to mediate DNA repair synthesis in permeabilized diploid human fibroblasts. However, it has now been suggested that in yeast, pol epsilon participates in DNA replication as well as DNA repair. However, while the "core" pol epsilon from yeast and HeLa have similar catalytic, 260 kDa subunits, they have unrelated accessory subunits. To understand the role of pol epsilon in mammalian cells, this laboratory very recently produced a panel of monoclonal antibodies to HeLa pol epsilon. In so doing, subsets of monoclonal antibodies to p260, the other "core" subunit, p55, and also to peptides which were shown to complex pol epsilon (p85, p70, p49) were obtained. p85 and p70 were identified as Ku autoantigen, a protein implicated in recombination and recombination repair. Using the antibodies, p55 will be sequenced, cloned and over-expressed jointly with p260. p49 will be identified and cloned. The significance of pol epsilon being complexed to Ku protein will be studied and whether the p350 protein kinase (which forms the Ku-associated DNA dependent protein kinase) also complexes to pol epsilon will be ascertained. From these results it is hoped to learn whether and how pol epsilon might be involved in a stress-response to DNA damage (e.g., might it monitor DNA during replication and call for a response, or might it be recruited for repair by such agents RNA pol II?). The monoclonal antibodies will also be used collaboratively to discern roles of human pol epsilon in nucleotide excision repair, recombinational repair, mismatch repair, replication and checkpoint control. A replication bypass assay will determine a possible role for pol epsilon in damage bypass replication. Finally a novel, extramitochondrial HeLa DNA polymerase with properties similar to both pols beta and gamma will be characterized with special attention to whether it is a homologue of yeast pol beta or a nuclear form of pol gamma.