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

古生物在数十亿年前进化为生命的第三个领域，但它们是生物体宇宙树中相对较新的成员。古生物显示出来自细菌和真核生物的镶嵌体，但它们也具有独特的分子特征。嗜热古菌是在80 ℃和更高温度下生长的超级微生物，并表现出不同寻常的特性，这使得这些生物体对于开发新的生物技术应用很有价值，但对于研究极端生命也很有趣。嗜热古菌对生命起源的研究也很重要，最近发现的洛基古菌群表明真核生物可能起源于古菌。尽管在过去的40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，关于古菌中DNA分离过程的信息很少，这一主题仍然是一个等待调查的黑匣子。基因分离是每个细胞生命周期中的一个关键阶段：遗传物质首先被复制，然后被分离并平均分配到子细胞中。我们最近研究了来自酸性温泉的硫化叶菌属物种中低拷贝数质粒所携带的分配系统的分子机制（Science：349：1120-1124）。这个质粒稳定遗传的工具包是一个三组分的机器，显示了与细菌和真核蛋白质的联系。该系统编码一个Walker型帕拉A、一个嵌合衔接子ParB和一个着丝粒结合因子AspA。AspA蛋白在DNA上扩散，产生螺旋对接平台，ParB N-末端结构域亚基在其上组装成第二个超螺旋。令人惊讶的是，ParB的C-末端表现出类似于CenpA histonevariant的结构折叠，其参与真核细胞中动粒的组装。这种独特的多蛋白质结构融合了原核和真核元素，表明DNA分离原则在生命的三个领域中保持不变。该项目旨在研究AspA-ParBParA多蛋白复合物在DNA上的组装和扩展过程，使用的工具包括DNA酶足迹和迁移率变动分析以及染色质免疫沉淀（ChIP-Seq），原子力显微镜（AFM）和微尺度热泳（MST）。ParB C末端CenpA样结构域与可能参与ParB转录后修饰的蛋白质的相互作用也将通过使用一个andem亲和纯化（TAP）方法来检查。另一个目的是对宿主硫化叶菌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 theyare a relatively recent addition to the universal tree of living organisms.Archaea show a mosaic of tesserae from bacteria and eukaryotes, butthey are also characterized by unique molecular features. Thermophilicarchaea are super microbes thriving at 80C and higher temperaturesand exhibiting unusual properties, which make these organisms valuablefor the development of novel biotechnological applications, but alsointeresting for studies on life pushed to extremes. Thermophilic archaeaare also important for studies on the origin of life and the recentdiscovery of the Lokiarchaeota group has suggested that eukaryotesmight have originated from archaea.Despite the significant progress made in decoding molecularmechanisms in these organisms in the last four decades, to date littleinformation is available on the process of DNA segregation in archaeaand the subject remains a black box awaiting investigation. Genomesegregation is a crucial stage of the life cycle of every cell: the geneticmaterial is first duplicated, then separated and equally distributed intodaughter cells. We have recently investigated the molecular machinery ofthe partition system harboured by a low copy number plasmid in aSulfolobus species from acidic hot springs (Science: 349: 1120-1124). Thetoolkit for the stable inheritance of this plasmid is a three-componentmachine showing linkages to bacterial and eukaryotic proteins. Thissystem encodes a Walker-type ParA, a chimaeric adaptor ParB and acentromere-binding factor, AspA. The AspA protein spreads on the DNAgenerating a helical docking platform onto which ParB N-terminusdomain subunits assemble into a second superhelix. Surprisingly, theParB C-terminus exhibits a structural fold similar to the CenpA histonevariant, which is involved in assembly of the kinetochore in eukaryoticcells. This unique multi-protein structure merges prokaryotic andeukaryotic elements, suggesting the conservation of DNA segregationprinciples across the three domains of life. The project aims toinvestigate the process of assembly and spreading of the AspA-ParBParAmulti-protein complex on the DNA by using tools such as DNasefootprinting and mobility shift assays as well as chromatinimmunoprecipitation (ChIP-Seq), atomic force microscopy (AFM) andmicroscale thermophoresis (MST). The interaction of the ParB CterminusCenpA-like domain with proteins potentially involved in posttranscriptionalmodification of ParB will also be examined by using atandem affinity purification (TAP) approach. An additional objective issequencing the chromosome of the host Sulfolobus strain to establishwhether there is a dynamic flux between chromosome and plasmid.