RECOGNITION SEQUENCE OF A HIGHLY CONSERVED DNA-BINDING PROTEIN RBP-JKAPPA
RECOGNITION SEQUENCE OF A HIGHLY CONSERVED DNA-BINDING PROTEIN RBP-JKAPPA
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DOI：
10.1093/nar/22.6.965
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发表时间：
1994-03-25
影响因子：
14.9
通讯作者：
KAWAICHI, M
中科院分区：
文献类型：
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作者：
TUN, T;HAMAGUCHI, Y;KAWAICHI, M
广泛检查RBP-J κ蛋白的DNA结合特异性。小鼠RBP-J κ蛋白最初分离为与J κ V（D）J型重组信号序列结合的核蛋白，所述重组信号序列由被23个碱基对间隔区隔开的保守七聚体（CACTGTG）和九聚体（GGTTTTTGT）序列组成。使用在J κ重组信号序列的各个部分中具有突变的DNA探针的电泳迁移率变动测定表明，RBP-J κ蛋白除了识别七聚体之外还识别重组信号之外的序列，但根本不识别九聚体序列和间隔子长度。数据库检索确定了果蝇分裂基因簇增强子中m8基因启动子区RBP-J κ蛋白的最佳天然存在的结合基序（CACTGTGGGAACGG）。与一系列的m8基序突变体的结合试验表明，该蛋白主要识别的GTGGGAA序列，也与ACT和CG序列侧翼这七核苷酸弱相互作用。通过重复电泳迁移率变动分析，从含有20个碱基随机序列的合成寡核苷酸池中富集与RBP-J κ蛋白结合的寡核苷酸。富集的寡聚体共享CGTGGGAA的共同序列。所有这些数据表明RBP-J κ蛋白识别CGTGGGAA的独特核心序列，并且不结合没有侧翼序列的V（D）J重组信号。
DNA binding specificity of the RBP-J kappa protein was extensively examined. The mouse RBP-J kappa protein was originally isolated as a nuclear protein binding to the J kappa type V(D)J recombination signal sequence which consisted of the conserved heptamer (CACTGTG) and nonamer (GGTTTTTGT) sequences separated by a 23-base pair spacer. Electrophoretic mobility shift assay using DNA probes with mutations in various parts of the J kappa recombination signal sequence showed that the RBP-J kappa protein recognized the sequence outside the recombination signal in addition to the heptamer but did not recognize the nonamer sequence and the spacer length at all. Database search identified the best naturally occurring binding motif (CACTGTGGGAACGG) for the RBP-J kappa protein in the promoter region of the m8 gene in the Enhancer of split gene cluster of Drosophila. The binding assay with a series of m8 motif mutants indicated that the protein recognized mostly the GTGGGAA sequence and also interacted weakly with ACT and CG sequences flanking this heptanucleotide. Oligonucleotides binding to the RBP-J kappa protein were enriched from a pool of synthetic oligonucleotides containing 20-base random sequences by the repeated electrophoretic mobility shift assay. The enriched oligomer shared a common sequence of CGTGGGAA. All these data indicate that the RBP-J kappa protein recognizes a unique core sequence of CGTGGGAA and does not bind to the V(D)J recombination signal without the flanking sequence.