Intercollegiate Faculty of Biotechnology of University of Gdansk and Medical University of Gdansk, University of Gdansk, Abrahama 58, 80-307 Gdansk, Poland.
Department of Physical Chemistry, Gdańsk University of Technology, Narutowicza 11/12, 80-233 Gdańsk, Poland.
Nucleic Acids Res. 2021 Apr 6;49(6):3394-3408. doi: 10.1093/nar/gkab113.
An essential feature of replication initiation proteins is their ability to bind to DNA. In this work, we describe a new domain that contributes to a replication initiator sequence-specific interaction with DNA. Applying biochemical assays and structure prediction methods coupled with DNA-protein crosslinking, mass spectrometry, and construction and analysis of mutant proteins, we identified that the replication initiator of the broad host range plasmid RK2, in addition to two winged helix domains, contains a third DNA-binding domain. The phylogenetic analysis revealed that the composition of this unique domain is typical within the described TrfA-like protein family. Both in vitro and in vivo experiments involving the constructed TrfA mutant proteins showed that the newly identified domain is essential for the formation of the protein complex with DNA, contributes to the avidity for interaction with DNA, and the replication activity of the initiator. The analysis of mutant proteins, each containing a single substitution, showed that each of the three domains composing TrfA is essential for the formation of the protein complex with DNA. Furthermore, the new domain, along with the winged helix domains, contributes to the sequence specificity of replication initiator interaction within the plasmid replication origin.
复制起始蛋白的一个重要特征是它们能够与 DNA 结合。在这项工作中，我们描述了一个新的结构域，该结构域有助于复制起始序列与 DNA 的特异性相互作用。通过应用生化测定、结构预测方法以及 DNA-蛋白交联、质谱分析和突变蛋白的构建和分析，我们确定了广泛宿主范围质粒 RK2 的复制起始蛋白除了两个翼状螺旋结构域外，还包含第三个 DNA 结合结构域。系统发育分析表明，该独特结构域的组成在描述的 TrfA 样蛋白家族中是典型的。涉及构建的 TrfA 突变蛋白的体外和体内实验表明，新鉴定的结构域对于与 DNA 形成蛋白复合物、增加与 DNA 的亲和力以及起始蛋白的复制活性都是必需的。对每个突变蛋白只包含一个取代的分析表明，构成 TrfA 的三个结构域中的每一个对于与 DNA 形成蛋白复合物都是必需的。此外，新结构域与翼状螺旋结构域一起有助于质粒复制起点内复制起始因子相互作用的序列特异性。
