FUNCTION OF HUMAN DAMAGED DNA BINDING PROTEIN (DDB)

描述(摘自申请者摘要)：这是补助金的修订版 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是一个 异源二聚体p127和p48的损伤偏好与E. ColiUvrA蛋白。此外，P48中的错义突变仅存在于 E组着色性干皮病患者(XP-E DDB-)的一个亚组，其 蛋白质缺乏活性。纯化的正常蛋白可纠正XP修复 仅当注射入DDB-细胞时才有缺陷。作为对DNA损伤的反应，P48但是 Not p127表达增加。最后，林恩博士和他最近的观察 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。 林博士现在建议探索单独的DDB亚单位在 DNA暴露后DNA修复和/或转录模式的变化 破坏性因素。特别是，他建议识别细胞成分 P127和p48与之相互作用。为了实现这一目标，他提议 构建缺乏p48或p127的转基因小鼠(或仅当 这些老鼠是不能存活的)。他将在昆虫中单独过度表达每个亚基 用细胞制作抗体和/或亲和柱来确定哪种蛋白质(S) 这些亚单位相互作用。除了抗体或DDB亚单位作为 亲和柱配体、凝集素可能是DDB糖基化的特异性 被利用了。林恩博士还将研究酵母二号和三号杂交筛选和启动子 进行排序以实现相同的目标。他已经制造了绿色荧光蛋白 含有p48和p127的杂交蛋白，以及抗体，将是 用于研究这些蛋白质的细胞定位和共定位 在正常细胞和XP-E细胞中都有可能与DDB相互作用的蛋白。全 相互作用将作为DNA损伤暴露和细胞的函数进行研究 周而复始。他将继续探索DDB互动的意义 与转录因子EBNA2和E2F1结合。最后，他希望获得 5‘非翻译区的序列数据，以寻找可以 提供多肽的功能建议。 庞氏沙门氏菌的基因组似乎编码人类p127的同源物，但不是 48页。此外，虽然S.pombe的提取物不含DDB活性，但 当将人P48添加到提取物中时，活性就会出现。林恩博士计划 使用遗传和生化分析来寻找这一现象的分子基础 P127基因的活性和定向突变的后果 同源。

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.