DNA helicases displace streptavidin from biotin-labeled oligonucleotides
DNA helicases displace streptavidin from biotin-labeled oligonucleotides
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DOI：
10.1021/bi9822269
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发表时间：
1999-04-20
期刊：
影响因子：
2.9
通讯作者：
Raney, KD
中科院分区：
文献类型：
--
作者：
Morris, PD;Raney, KD
解旋酶是利用三磷酸核苷水解产生的能量解开双链 (ds) DNA 的酶，这一过程几乎对 DNA 代谢的每个阶段都至关重要。本研究中使用的解旋酶 gp41 和 Dda 来自噬菌体 T4，这是研究处理 DNA 的酶的出色系统。 gp41 是复制解旋酶，已证明在 ATP 存在的情况下形成六聚体。在本研究中，在线性或环状单链 (ss) DNA 底物存在的情况下进行蛋白质交联，以确定 gp41 与 ssDNA 结合的拓扑结构。结果表明，六聚体通过环绕 ssDNA 来结合它，其方式与其他六聚体解旋酶类似。开发了一种新的测定方法来研究 gp41 和 Dda 对单链 DNA 的酶活性。在解旋酶存在下，通过电泳迁移率变动测定法测定链霉亲和素从各种生物素化寡核苷酸的解离速率。发现 gp41 和 Dda 在 ATP 依赖性反应中显着提高链霉亲和素与生物素标记寡核苷酸的解离速率。解旋酶催化链霉亲和素与生物素标记的 62 聚体寡核苷酸 3' 端的解离发生，一级速率为 0.17 min(-1)，比生物素与链霉亲和素的自发解离速率快 500 倍以上。 Dda 活性导致链霉亲和素从 62 聚体 3' 端更快地位移，一级速率为 7.9 s(-1)。这比自发解离率高一百万倍以上。任一解旋酶均未增强链霉亲和素与 5'-生物素标记的寡核苷酸的解离。每个解旋酶都能够将链霉亲和素从 3'-生物素标签上移开，这一事实表明这些酶能够对阻断其路径的分子施加力。寡核苷酸 5' 和 3' 末端之间的位移差异也与每个解旋酶 ssDNA 上易位的 5' 至 3' 方向偏差的可能性一致。
Helicases are enzymes that use energy derived from nucleoside triphosphate hydrolysis to unwind double-stranded (ds) DNA, a process vital to virtually every phase of DNA metabolism. The helicases used in this study, gp41 and Dda, are fi om the bacteriophage T4, an excellent system for studying enzymes that process DNA. gp41 is the replicative helicase and has been shown to form a hexamer in the presence of ATP. In this study, protein cross-linking was performed in the presence of either linear or circular single-stranded (ss) DNA substrates to determine the topology of gp41 binding to ssDNA. Results indicate that the hexamer binds ssDNA by encircling it, in a manner similar to that of other hexameric helicases. A new assay was developed for studying enzymatic activity of gp41 and Dda on single-stranded DNA. The rate of dissociation of streptavidin from various biotinylated oligonucleotides was determined in the presence of helicase by an electrophoretic mobility shift assay. gp41 and Dda were found to significantly enhance the dissociation rate of streptavidin from biotin-labeled oligonucleotides in an ATP-dependent reaction. Helicase-catalyzed dissociation of streptavidin from the 3'-end of a biotin-labeled 62-mer oligonucleotide occurred with a first-order rate of 0.17 min(-1) which is over 500-fold faster than the spontaneous dissociation rate of biotin from streptavidin. Dda activity leads to even faster displacement of streptavidin from the 3' end of the 62-mer, with a first-order rate of 7.9 s(-1). This is more than a million-fold greater than the spontaneous dissociation rate. There was no enhancement of streptavidin dissociation from the 5'-biotin-labeled oligonucleotide by either helicase. The fact that each helicase was capable of dislodging streptavidin from the 3'-biotin label suggests that these enzymes are capable of imparting a force on a molecule blocking their path. The difference in displacement between the 5' and 3' ends of the oligonucleotide is also consistent with the possibility of a 5'-to-3' directional bias in translocation on ssDNA for each helicase.