Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan.
Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 300, Taiwan.
Nucleic Acids Res. 2024 Sep 9;52(16):9966-9977. doi: 10.1093/nar/gkae660.
Genome segregation is a fundamental process that preserves the genetic integrity of all organisms, but the mechanisms driving genome segregation in archaea remain enigmatic. This study delved into the unknown function of SegC (SSO0033), a novel protein thought to be involved in chromosome segregation in archaea. Using fluorescence polarization DNA binding assays, we discovered the ability of SegC to bind DNA without any sequence preference. Furthermore, we determined the crystal structure of SegC at 2.8 Å resolution, revealing the multimeric configuration and forming a large positively charged surface that can bind DNA. SegC has a tertiary structure folding similar to those of the ThDP-binding fold superfamily, but SegC shares only 5-15% sequence identity with those proteins. Unexpectedly, we found that SegC has nucleotide triphosphatase (NTPase) activity. We also determined the SegC-ADP complex structure, identifying the NTP binding pocket and relative SegC residues involved in the interaction. Interestingly, images from negative-stain electron microscopy revealed that SegC forms filamentous structures in the presence of DNA and NTPs. Further, more uniform and larger SegC-filaments are observed, when SegA-ATP was added. Notably, the introduction of SegB disrupts these oligomers, with ATP being essential for regulating filament formation. These findings provide insights into the functional and structural role of SegC in archaeal chromosome segregation.
基因组分离是一个维持所有生物体遗传完整性的基本过程，但驱动古菌基因组分离的机制仍然是个谜。本研究深入研究了 SegC（SSO0033）的未知功能，SegC 是一种新型蛋白，被认为参与古菌的染色体分离。我们使用荧光偏振 DNA 结合测定法发现，SegC 具有结合 DNA 的能力，而没有任何序列偏好。此外，我们确定了 SegC 的晶体结构，分辨率为 2.8Å，揭示了多聚体构型，并形成了一个可以结合 DNA 的大的正电荷表面。SegC 的三级结构折叠类似于 ThDP 结合折叠超家族，但 SegC 与这些蛋白的序列同一性仅为 5-15%。出乎意料的是，我们发现 SegC 具有核苷酸三磷酸酶（NTPase）活性。我们还确定了 SegC-ADP 复合物结构，确定了 NTP 结合口袋和参与相互作用的相对 SegC 残基。有趣的是，负染电子显微镜图像显示，在 DNA 和 NTPs 的存在下，SegC 形成丝状结构。进一步观察到，当添加 SegA-ATP 时，SegC 形成更均匀和更大的丝状结构。值得注意的是，SegB 的引入会破坏这些寡聚物，而 ATP 对于调节丝状结构的形成是必不可少的。这些发现为 SegC 在古菌染色体分离中的功能和结构作用提供了新的见解。
