FUNCTION OF HUMAN DAMAGED DNA BINDING PROTEIN (DDB)

描述（来自申请人的摘要）：本授权的修订版 申请是一项继续研究人类损伤DNA结合的建议 蛋白质（DDB）。在以前的研究中，申请人已经证明DDB是一种 p127和p48的异源二聚体，其具有与E. coliUvrA蛋白。此外，p48的错义突变存在于-且仅存在于 在着色性干皮病E组患者（XP-E Ddb-）的一个亚组中， 蛋白质缺乏活性。纯化的正常蛋白可以纠正XP修复 只有当注入Ddb-细胞时才有缺陷。为了应对DNA损伤，p48， 而不是p127表达增加。最后，Linn博士最近的观察和 其他研究表明，DDB与几种细胞和病毒转录相互作用， 反式激活蛋白 Linn博士现在建议探索单个DDB亚基在以下方面的作用： 暴露于DNA后DNA修复和/或转录模式的变化 破坏剂。特别是，他建议识别细胞成分， 与p127和p48相互作用。为了实现这一目标，他建议 构建缺乏p48或p127的反式杂交小鼠（或仅当 小鼠不能存活）。他将在昆虫细胞中单独过表达每个亚基， 细胞来制备抗体和/或亲和柱来确定哪种蛋白质 这些亚单位相互作用。除了抗体或DDB亚基， 亲和柱配体，对DDB糖基化特异性的凝集素可以是 利用。Linn博士还将做酵母二和三杂交筛选和启动子 为了实现同一个目标。他制造了绿色荧光蛋白 与p48和p127杂交的蛋白质，以及这些蛋白质，沿着抗体，将被 用来观察这些蛋白质的细胞定位和共定位 与正常细胞和XP-E细胞中任何假定的DDB相互作用蛋白质。所有 将研究相互作用作为DNA损伤暴露和细胞 周期他将继续探讨DDB互动的意义 转录因子EBNA 2和E2 F1。最后，他希望获得 5'非翻译区的序列数据，以寻找可以 为肽的功能提供建议。 S.粟酒裂殖酵母似乎编码人类p127的同源物，但不编码人类p127的同源物。 第48页。此外，S.粟酒不含DDB活性，例如 在向提取物中加入人p48后出现活性。林博士计划 使用遗传和生物化学分析来找到这一点的分子基础。 活性和假定的p127中定向突变的后果 同源物

DESCRIPTION (from applicant's abstract): This revised version of the grant application is a proposal to continue studies of human Damage DNA Binding Protein (DDB). In previous studies, the applicant has shown DDB to be a heterodimer of p127 and p48 which has roughly the same damage preference as E. coli UvrA protein. In addition, missense mutations in p48 exist in - and only in - a subset of Xeroderma pigmentosum group E patients (XP-E Ddb-) whose protein lacks activity. Purified normal protein can correct the XP repair defect only when injected into Ddb- cells. In response to DNA damage, p48 but not p127 expression is increased. Finally, recent observations by Dr. Linn and others show that DDB interacts with several cellular and viral transcription transactivating proteins. Dr. Linn now proposes to explore the role(s) of the individual DDB subunits in DNA repair and/or transcription pattern changes following exposures to DNA damaging agents. In particular, he proposes to identify cellular components with which p127 and p48 interact. To accomplish this goal, he proposes to construct trans-hybrid mice lacking either p48 or p127 (or cell lines only if the mice are unviable). He will overexpress each subunit individually in insect cells to make antibodies and/or affinity columns to determine which protein(s) the subunits interact with. In addition to antibodies or DDB subunits as affinity column ligands, lectins specific for the DDB glycosylation may be utilized. Dr. Linn will also do yeast two and three hybrid screens and promoter sequencing to accomplish the same goal. He has made green fluorescent protein hybrid proteins with p48 and p127 and these, along with antibodies, will be used to look at cellular localization of these proteins and co-localization with any putative DDB-interacting proteins in both normal and XP-E cells. All interactions will be studied as a function of DNA damage exposure and cell cycle. He will continue to explore the significance of the interaction of DDB with the transcription factors EBNA2 and E2F1. Finally, he expects to obtain sequence data of the 5' un-translated region to look for motifs which could provide suggestions of functions for the peptides. The genome of S. pombe appears to code for a homologue of human p127 but not of p48. Moreover, while extracts of S. pombe do not contain DDB activity, such activity appears upon addition of human p48 to the extracts. Dr. Linn plans to use both genetic and biochemical analysis to find the molecular basis of this activity and the consequences of directed mutations in the putative p127 homologue.