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

古生菌在数十亿年前演化为生命的第三个领域，但它们是相对较新的生物物种。古生菌显示了细菌和真核生物的镶嵌，但它们也具有独特的分子特征。嗜热古生菌是一种超级微生物，在80摄氏度和更高的温度下茁壮成长，并表现出不同寻常的性质，这使得这些生物对于开发新的生物技术应用非常有价值，但对于被推向极端的生命研究也很有趣。嗜热考古对于生命起源的研究也很重要，最近Loki考古群的发现表明真核生物可能起源于考古。尽管在过去40年里在破译这些生物的分子机制方面取得了重大进展，但到目前为止，关于考古中DNA分离过程的信息很少，这个课题仍然是一个等待研究的黑匣子。基因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，然后分离并平均分配到子代细胞中。我们最近研究了来自酸性温泉的硫磺中含有低拷贝数质粒的分配系统的分子机制(Science：349：1120-1124)。该质粒稳定遗传的试剂盒是一个三组分的机器，显示了与细菌和真核蛋白的联系。该系统编码Walker-type parA、嵌合接头PARB和无着丝粒结合因子ASPA。ASPA蛋白在DNA上扩散，产生一个螺旋对接平台，PARB N-末端结构域亚单位在该平台上组装成第二个超螺旋。令人惊讶的是，ParB的C末端显示了类似于CenpA组变体的结构折叠，CenpA组变体参与了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生物和真核生物的成分，表明生命的三个领域的DNA分离原则是保守的。该项目旨在利用DNA足迹和迁移率改变分析等工具，以及免疫沉淀(CHIP-SEQ)、原子力显微镜(AFM)和微尺度热电泳法(MST)等工具，研究ASPA-ParBParA多蛋白复合体在DNA上的组装和铺展过程。还将用串联亲和纯化(TAP)的方法研究PARB CTerusCenpA样结构域与可能参与PARB转录后修饰的蛋白质的相互作用。另一个目标是对宿主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流动。

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.