In vitro selection of DNA binding sites for ABF1 protein from Saccharomyces cerevisiae
In vitro selection of DNA binding sites for ABF1 protein from Saccharomyces cerevisiae
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DOI：
10.1016/j.febslet.2005.07.009
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发表时间：
2005-08-15
期刊：
影响因子：
3.5
通讯作者：
Krauss, G
中科院分区：
文献类型：
--
作者：
Beinoraviciute-Kellner, R;Lipps, G;Krauss, G
酿酒酵母(Sacchramoyces Cerevisiae)自主复制序列结合因子I(ABFI)是一种多功能的DNA结合蛋白，参与转录调控、DNA复制以及通过核小体重塑重组染色质。ABFI与ARS元件和各种转录调节元件中的DNA序列结合。这导致了对协商一致动机5‘--CGTnnnnGA(G/C)-3’的早期定义。我们使用了SELEX方法来扩展和更好地描述ABFI的DNA序列要求。从随机排列的30个核苷酸序列的寡核苷酸池开始，我们使用EMSA来选择与ABFI具有高亲和力的序列。在第15轮SELEX后，我们获得了106个适配子的序列。通过与Motif搜索程序Meme的分析，从这个库中获得了16个核苷酸的共识。定量EMSA实验验证了我们的实验方法，因为高亲和力结合的结合序列在池中出现的频率更高，并在更高程度上与推导出的共识相似。我们发现了与ABFI结合的DNA序列，与迄今接受的ABFI一致序列相比，亲和力几乎高出两个数量级。这导致我们假设了一个强烈的识别动机：5‘-TnnCGTnnnnnT-GAT-3’。(C)2005年欧洲生化学会联合会。爱思唯尔出版，版权所有。
The autonomously replicating sequence-binding factor I (ABFI) from Sacchramoyces cerevisiae is known as a multifunctional DNA binding protein that is involved in transcriptional regulation, DNA-replication, and in restructuring of chromatin via nucleosome remodelling. ABFI binds to DNA sequences found in ARS elements and in various transcriptional regulatory elements. This led to the early definition of the consensus motive 5'-CGTnnnnnGA(G/C)-3'. We have used a SELEX approach to expand and better characterize the DNA sequence requirements of ABFI. Starting from a pool of oligonucleotides randomized at a sequence of 30 nucleotides, we used EMSA to select for sequences with high affinity for ABFI. We obtained the sequences of 106 aptamers after the 15th SELEX round. A 16 nucleotide consensus was derived from this pool by analysis with the motif search programme MEME. Quantitative EMSA experiments verified our experimental approach since binding sequences which were bound with high affinity occurred more often in the pool and resembled the derived consensus to a higher degree. We found DNA sequences that are bound by ABFI with nearly two-magnitude higher affinity as compared to the hitherto accepted ABFI consensus sequence. This led us to postulate a strong recognition motive: 5'-TnnCGTnnnnnnT-GAT-3'. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.