DNA POLYMERASES

描述：由于DNA序列的变化具有深刻的遗传学意义 后果，重要的是要了解维持 如果我们要完全理解这些序列的基础，复制这些序列 遗传性和退化性疾病及其治疗的可能目标。 DNA聚合酶显然是DNA新陈代谢的关键角色，它是 申请者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，该小组鉴定并纯化了一种DNA聚合酶(POL Epsilon)，用于介导渗透性DNA修复合成 二倍体人成纤维细胞。然而，现在有人建议，在 酵母、Polepsilon参与DNA复制和DNA修复。 然而，尽管酵母和HeLa的“核心”Polepsilon具有相似的 催化，260 kDa亚基，它们有无关的附属亚基。至 了解Polepsilon在哺乳动物细胞中的作用，这个实验室非常 最近研制出了一组抗HeLa polepsilon的单抗。在……里面 如此一来，抗p260的单抗的亚群，另一个“核心” 亚基，P55，以及显示为复杂Polepsilon的多肽 (P85、P70、P49)。P85和p70被鉴定为Ku 自身抗原，一种与重组和重组修复有关的蛋白质。 利用这些抗体，将对p55进行测序、克隆和过度表达 与P260联合使用。将对p49进行鉴定和克隆。的重要意义。 将研究POL epsilon与Ku蛋白的络合以及是否 P350蛋白激酶(形成Ku相关的DNA依赖蛋白 此外，还将确定Polepsilon的络合物。从这些 结果希望了解Polepsilon是否以及如何参与其中。 在对DNA损伤的应激反应中(例如，它是否可以在 复制并呼吁做出回应，或者它可能会被招募进行修复 这类药物是RNA PolII？)。 这些单抗也将被联合使用来辨别角色 人类Polepsilon在核苷酸切除修复，重组修复， 错配修复、复制和检查点控制。绕过复制 化验将确定Polepsilon在损伤旁路中的可能作用 复制。 最后得到一种新的线粒体外HeLa DNA聚合酶 与Pols类似，Beta和Gamma都将具有特殊的特性 注意它是否是酵母POLβ的同系物或核形式 波尔伽马。

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.