FUNCTION OF HUMAN DAMAGED DNA BINDING PROTEIN (DDB)

人体受损 DNA 结合蛋白 (DDB) 的功能

• 批准号: 6386493
• 负责人: STUART M LINN
• 金额: $ 33.84万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2004-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386493
• 关键词: DNA binding protein; DNA damage; DNA repair; Schizosaccharomyces pombe; gene mutation; gene targeting; genetic promoter element; genetic transcription; genetically modified animals; green fluorescent proteins; human genetic material tag; laboratory mouse; nucleic acid sequence; protein protein interaction; protein structure function; yeast two hybrid system

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.

描述（摘自申请人的摘要）：该赠款的修订版 应用是继续研究人类损伤DNA结合的建议 蛋白质（DDB）。在先前的研究中，申请人已显示DDB为 p127和p48的异二聚体，其损伤偏好大致相同。 大肠杆菌蛋白。此外，p48中的错义突变存在于 - 只有 在 - 静脉表色素组E组（XP-E DDB-）的子集中 蛋白质缺乏活性。纯化的正常蛋白可以纠正XP修复 仅当注入DDB细胞时缺陷。响应DNA损伤，P48，但 不是P127表达增加。最后，Linn博士和 其他表明DDB与几个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亚基的作用 暴露于DNA后的DNA修复和/或转录模式变化 有害代理。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，他建议 构建缺乏P48或P127的跨杂交小鼠（或仅在细胞系 小鼠是不可行的）。他将在昆虫中分别过表达每个亚基 细胞制作抗体和/或亲和力柱以确定​​哪种蛋白 亚基与。除抗体或DDB亚基外 亲和力柱配体，特异性DDB糖基化的凝集素可能是 利用。 Linn博士还将制作酵母二和三的混合动力屏幕和启动子 测序以实现相同的目标。他制作了绿色荧光蛋白 带有p48和p127的混合蛋白，这些蛋白以及抗体将是 用于查看这些蛋白质的细胞定位和共定位 与正常和XP-E细胞中的任何假定的DDB相互作用蛋白质。全部 相互作用将作为DNA损伤暴露和细胞的函数研究 循环。他将继续探索DDB相互作用的重要性 与转录因子EBNA2和E2F1。最后，他希望获得 5'未翻译区域的序列数据寻找可能 提供肽功能的建议。 S. pombe的基因组似乎是为人类P127的同源物编码 P48。此外，而S. pombe的提取物不包含DDB活动，例如 在将人p48添加到提取物中后会出现活性。 Linn博士计划 同时使用遗传和生化分析来找到这一点的分子基础 推定P127中的活动和定向突变的后果 同源物。