Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic.
J Biomol Struct Dyn. 2012;30(4):371-8. doi: 10.1080/07391102.2012.682205.
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 14-3-3γ protein binds strongly to cruciform structures and is crucial for initiating replication. In this study, we analyzed DNA binding properties of the 14-3-3γ isoform to linear and supercoiled DNA. We demonstrate that 14-3-3γ protein binds strongly to long DNA targets, as evidenced by electrophoretic mobility shift assay on agarose gels. Binding of 14-3-3γ 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 14-3-3γ protein in its binding to local DNA structures which are stabilized by DNA supercoiling.
14-3-3 蛋白家族是一组高度保守且广泛分布的蛋白，由真核生物中的多个同工型组成。14-3-3 蛋白在细胞中普遍表达，在 DNA 复制、细胞周期调控和细胞凋亡中发挥关键作用。14-3-3 蛋白的功能通过与大量其他蛋白和 DNA 的相互作用来介导。已经证明 14-3-3γ 蛋白与十字形结构强烈结合，对于起始复制至关重要。在本研究中，我们分析了 14-3-3γ 同工型与线性和超螺旋 DNA 的 DNA 结合特性。我们证明 14-3-3γ 蛋白强烈结合长 DNA 靶标，这一点通过琼脂糖凝胶上的电泳迁移率变动分析得到证实。14-3-3γ 与 DNA 靶标的结合导致模糊、延迟的 DNA 带出现。在线性和超螺旋 DNA 上的竞争实验表明对超螺旋 DNA 具有非常强的偏好。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 14-3-3γ 蛋白在其与局部 DNA 结构的结合中起作用，这些局部 DNA 结构通过 DNA 超螺旋稳定。
