Borrowing building blocks from bacteria and eukaryotes: a three-component DNA segregation machinery in archaea

古生菌是在数十亿年前作为生命的第三个领域进化而来的，但它们是在宇宙生物树中相对较新的成员。古生菌是细菌和真核生物的镶嵌体，但它们也具有独特的分子特征。嗜热古细菌是一种超级微生物，在80摄氏度及更高的温度下繁殖，并表现出不同寻常的特性，这使得这些生物对开发新的生物技术应用有价值，但对极端生命的研究也很有趣。嗜热古细菌对生命起源的研究也很重要，最近发现的Lokiarchaeota群表明真核生物可能起源于古细菌。尽管在过去的四十年里，在解码这些生物的分子机制方面取得了重大进展，但迄今为止，关于古细菌DNA分离过程的信息很少，这个主题仍然是一个等待调查的黑盒子。基因组分离是每个细胞生命周期的关键阶段：遗传物质首先被复制，然后被分离并均匀分布到子细胞中。我们最近研究了酸性温泉中Sulfolobus物种的低拷贝数质粒所携带的分区系统的分子机制（Science: 349: 1120-1124）。该质粒稳定遗传的工具箱是一个三组分机器，显示了与细菌和真核蛋白的联系。该系统编码一个walker型ParA，一个嵌合接头ParB和一个着丝粒结合因子AspA。AspA蛋白在DNA上扩散，形成一个螺旋对接平台，在这个平台上，ParB n端结构域亚基组装成第二个超螺旋。令人惊讶的是，ParB的c端显示出与CenpA组蛋白变体相似的结构折叠，这与真核细胞中着丝点的组装有关。这种独特的多蛋白结构融合了原核和真核元素，表明在生命的三个领域中DNA分离原则是守恒的。该项目旨在研究AspA-ParB- ParA多蛋白复合物在DNA上的组装和扩散过程，方法包括DNA酶足迹测定和迁移位移测定，以及染色质免疫沉淀（ChIP-Seq）、原子力显微镜（AFM）和微尺度热泳法（MST）。ParB C末端CenpA-like结构域与可能参与ParB转录后修饰的蛋白质的相互作用也将通过串联亲和纯化（TAP）方法进行研究。另一个目的是对宿主Sulfolobus菌株的染色体进行测序，以确定染色体和质粒之间是否存在动态通量。

Archaea evolved as the third domain of life billions of years ago, but they are a relatively recent addition to the universal tree of living organisms. Archaea show a mosaic of tesserae from bacteria and eukaryotes, but they are also characterized by unique molecular features. Thermophilic archaea are super microbes thriving at 80oC and higher temperatures and exhibiting unusual properties, which make these organisms valuable for the development of novel biotechnological applications, but alsointeresting for studies on life pushed to extremes. Thermophilic archaea are also important for studies on the origin of life and the recent discovery of the Lokiarchaeota group has suggested that eukaryotes might have originated from archaea.Despite the significant progress made in decoding molecular mechanisms in these organisms in the last four decades, to date little information is available on the process of DNA segregation in archaea and the subject remains a black box awaiting investigation. Genome segregation is a crucial stage of the life cycle of every cell: the geneticmaterial is first duplicated, then separated and equally distributed into daughter cells. We have recently investigated the molecular machinery of the partition system harboured by a low copy number plasmid in a Sulfolobus species from acidic hot springs (Science: 349: 1120-1124). The toolkit for the stable inheritance of this plasmid is a three-component machine showing linkages to bacterial and eukaryotic proteins. This system encodes a Walker-type ParA, a chimaeric adaptor ParB and a centromere-binding factor, AspA. The AspA protein spreads on the DNA generating a helical docking platform onto which ParB N-terminus domain subunits assemble into a second superhelix. Surprisingly, the ParB C-terminus exhibits a structural fold similar to the CenpA histone variant, which is involved in assembly of the kinetochore in eukaryoticcells. This unique multi-protein structure merges prokaryotic and eukaryotic elements, suggesting the conservation of DNA segregation principles across the three domains of life. The project aims to investigate the process of assembly and spreading of the AspA-ParB- ParA multi-protein complex on the DNA by using tools such as DNase footprinting and mobility shift assays as well as chromatin immunoprecipitation (ChIP-Seq), atomic force microscopy (AFM) and microscale thermophoresis (MST). The interaction of the ParB C- terminus CenpA-like domain with proteins potentially involved in post- transcriptional modification of ParB will also be examined by using a tandem affinity purification (TAP) approach. An additional objective is sequencing the chromosome of the host Sulfolobus strain to establish whether there is a dynamic flux between chromosome and plasmid.