Molecular evolution of DNA topoisomerase III beta (TOP3B) in Metazoa

DNA topoisomerase III beta (TOP3B) is unique because it acts on both DNA and RNA substrates to regulate gene expression and genomic stability. Mutations in human TOP3B are linked to neurodevelopmental and cognitive disorders, highlighting its relevance for human health. Despite the emerging importance of TOP3B, their precise cellular functions and evolutionary history remain poorly understood. Here we present the most comprehensive phylogenetic and protein structure comparative analyses of TOP3B. We confirmed that TOP3B is widespread and conserved across Metazoa. Subdomain IV was identified as the most conserved TOP3B region, in agreement with its role in providing the structural foundation for the protein. On the contrary, subdomain II was the less conserved, possibly by being the most structurally flexible of all TOP3B regions. Moreover, we identified a subset of TOP3B sites that interact with the TDRD3 auxiliary factor highly conserved among humans and other species. In contrast, TOP3B residue at position 472, previously associated with schizophrenia, is highly variable across animals, suggesting a more specific role in humans and related species. Finally, we showed that all TOP3B CXXC zinc finger motifs previously identified at the protein C-terminal region are retained across metazoans. We also found that the two major methylation sites known to regulate TOP3B activity are located in the most conserved section of the C-terminal arginine-glycine-glycine (RGG) box, suggesting that a similar regulatory mechanism may operate throughout metazoans. Overall, the data provided here lays the framework for a better understanding of the evolution and functional roles of TOP3B.

DNA拓扑异构酶IIIβ（DNA topoisomerase III beta, TOP3B）的独特之处在于可同时作用于DNA与RNA底物，以调控基因表达与基因组稳定性。人类TOP3B的突变与神经发育障碍及认知功能异常相关，凸显了其对人类健康的重要意义。尽管TOP3B的研究价值日益凸显，但其确切的细胞功能与进化历程仍鲜为人知。本研究针对TOP3B开展了迄今为止最为全面的系统发育与蛋白质结构比较分析，证实其在后生动物界（Metazoa）中广泛分布且高度保守。研究发现IV型亚结构域是TOP3B最为保守的区域，这与其为蛋白质提供结构基础的功能相符；与之相反，II型亚结构域的保守性最低，这可能源于其是所有TOP3B区域中结构柔性最强的部分。此外，本研究鉴定出一批可与TDRD3辅助因子相互作用的TOP3B位点，该类位点在人类及其他物种中均高度保守。与之形成对比的是，此前与精神分裂症相关的472号位置TOP3B氨基酸残基在动物界中变异度极高，提示其在人类及近缘物种中可能发挥更为特异的功能。最后，本研究证实，此前在蛋白质C端区域鉴定出的所有TOP3B CXXC锌指基序（CXXC zinc finger motif）在各后生动物中均得以保留。本研究还发现，已知的两个调控TOP3B活性的主要甲基化位点，均位于C端精氨酸-甘氨酸-甘氨酸（RGG）盒的最保守区域内，这提示类似的调控机制可能广泛存在于后生动物界中。综上，本研究提供的数据为深入解析TOP3B的进化历程与功能角色奠定了坚实的研究框架。