Huang Yen-Hua, Chen I-Chen, Huang Cheng-Yang
School of Biomedical Sciences, Chung Shan Medical University No. 110, Sec. 1, Chien-Kuo N. Rd. Taichung City Taiwan
Department of Medical Research, Chung Shan Medical University Hospital No. 110, Sec. 1, Chien-Kuo N. Rd. Taichung City Taiwan.
RSC Adv. 2019 Dec 5;9(69):40388-40396. doi: 10.1039/c9ra09406g. eCollection 2019 Dec 3.
Single-stranded DNA (ssDNA)-binding proteins (SSBs) play an important role in all DNA-dependent cellular processes, such as DNA replication, recombination, repair, and replication restart. The N-terminal domain of SSBs forms an oligonucleotide/oligosaccharide-binding (OB) fold for ssDNA binding. The SSB-dC35 complex structure has revealed how an SSB (EcSSB) tetramer binds to 65-nucleotide (nt)-long ssDNA, namely, the (SSB) binding mode. Knowledge on whether the ssDNA-binding mode for EcSSB is typical for all SSBs or is bacterial strain and length dependent is limited. Here, we studied the ssDNA-binding properties of a SSB (PaSSB) and investigated its interaction mode through crystallographic analysis. The complex crystal structure containing a PaSSB tetramer with two ssDNA chains was solved at a resolution of 1.91 Å (PDB entry 6IRQ). Results revealed that each bound ssDNA dT25 adopts an S-shaped conformation. This binding mode, as shown by the complex structure of PaSSB, differs significantly from (SSB). ssDNA-binding contributions from aromatic residues in PaSSB, except the contribution of Trp54, were not significant. Using electrophoretic mobility shift analysis, we characterized the stoichiometry of PaSSB complexed with a series of ssDNA homopolymers. The minimal length of ssDNA required for PaSSB tetramer binding and the size of the ssDNA-binding site were 25 and 29 nt, respectively. These observations through structure-function analysis suggested that only two OB folds rather than four OB folds in PaSSB are enough for the formation of a stable complex with ssDNA. The PaSSB noninteracting OB folds proposed here may allow sliding reptation in a dynamic ssDNA binding process.
单链DNA（ssDNA）结合蛋白（SSB）在所有依赖DNA的细胞过程中发挥着重要作用，如DNA复制、重组、修复和复制重启。SSB的N端结构域形成一个用于结合ssDNA的寡核苷酸/寡糖结合（OB）折叠。SSB-dC35复合物结构揭示了SSB（EcSSB）四聚体如何结合65个核苷酸（nt）长的ssDNA，即（SSB）结合模式。关于EcSSB的ssDNA结合模式是否对所有SSB都具有典型性，或者是否依赖于细菌菌株和长度的知识有限。在这里，我们研究了一种SSB（PaSSB）的ssDNA结合特性，并通过晶体学分析研究了其相互作用模式。以1.91 Å的分辨率解析了包含一个PaSSB四聚体与两条ssDNA链的复合物晶体结构（PDB条目6IRQ）。结果表明，每个结合的ssDNA dT25呈S形构象。如PaSSB的复合物结构所示，这种结合模式与（SSB）有显著差异。除了Trp54的贡献外，PaSSB中芳香族残基对ssDNA结合的贡献不显著。使用电泳迁移率变动分析，我们表征了PaSSB与一系列ssDNA同聚物复合的化学计量。PaSSB四聚体结合所需的ssDNA最小长度和ssDNA结合位点的大小分别为25 nt和29 nt。这些通过结构-功能分析的观察结果表明，PaSSB中只有两个OB折叠而不是四个OB折叠就足以与ssDNA形成稳定的复合物。这里提出的PaSSB非相互作用OB折叠可能允许在动态ssDNA结合过程中进行滑动爬行。
