Superhelical DNA as a preferential binding target of 14-3-3γ protein
Superhelical DNA as a preferential binding target of 14-3-3γ protein
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DOI：
10.1080/07391102.2012.682205
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发表时间：
2012-01-01
影响因子：
4.4
通讯作者：
Jagelska, Eva B.
中科院分区：
文献类型：
--
作者：
Brazda, Vaclav;Cechova, Jana;Jagelska, Eva B.
14-3-3蛋白家族是真核生物中高度保守且广泛分布的蛋白质组，由多种异构体组成。广泛表达的14-3-3蛋白在DNA复制、细胞周期调节和细胞凋亡中起关键作用。14-3-3蛋白的功能是通过与大量其他蛋白和DNA的相互作用来介导的。已经证明，14-3-3 γ蛋白与十字形结构强烈结合，并且对于启动复制至关重要。在这项研究中，我们分析了14-3-3 γ亚型与线性和超螺旋DNA的DNA结合特性。我们证明，14-3-3 γ蛋白强烈结合到长的DNA目标，证明了琼脂糖凝胶电泳迁移率变动分析。14-3-3 γ与DNA靶标的结合导致出现模糊、延迟的DNA条带。磁珠上线性和超螺旋DNA的竞争实验显示出对超螺旋DNA的非常强的偏好。我们还通过共聚焦显微镜显示，HCT-116细胞系中的14-3-3蛋白与DNA十字形共定位。这意味着14-3-3 γ蛋白在其与通过DNA超螺旋稳定的局部DNA结构的结合中的作用。
The 14-3-3 protein family is a highly conserved and widely distributed group of proteins consisting of multiple isoforms in eukaryotes. Ubiquitously expressed, 14-3-3 proteins play key roles in DNA replication, cell cycle regulation, and apoptosis. The function of 14-3-3 proteins is mediated by interaction with a large number of other proteins and with DNA. It has been demonstrated that 14-3-3 gamma protein binds strongly to cruciform structures and is crucial for initiating replication. In this study, we analyzed DNA binding properties of the 14-3-3 gamma isoform to linear and supercoiled DNA. We demonstrate that 14-3-3 gamma protein binds strongly to long DNA targets, as evidenced by electrophoretic mobility shift assay on agarose gels. Binding of 14-3-3 gamma to DNA target results in the appearance of blurry, retarded DNA bands. Competition experiments with linear and supercoiled DNA on magnetic beads show very strong preference for supercoiled DNA. We also show by confocal microscopy that 14-3-3 protein in the HCT-116 cell line is co-localized with DNA cruciforms. This implies a role for the 14-3-3 gamma protein in its binding to local DNA structures which are stabilized by DNA supercoiling.