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

借用细菌和真核生物的构建模块：古细菌中的三组分 DNA 分离机制

• 批准号: 1949055
• 金额: --
• 依托单位: University of York
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1949055
• 关键词: Borrowing; building; blocks; bacteria; eukaryotes

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.

古生物在数十亿年前进化为生命的第三个领域，但它们是生命有机体宇宙树中相对较新的成员。微孢子虫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 ℃和更高温度下蓬勃发展的超级微生物，并表现出不同寻常的特性，这使得这些生物体对开发新的生物技术应用很有价值，但也对极端生命的研究很感兴趣。嗜热古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菌。尽管在过去的40年里，对这些生物分子机制的解码取得了重大进展，但迄今为止，关于古菌DNA分离过程的信息仍然很少，这一主题仍然是一个有待研究的黑匣子。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，然后被分离并平均分配到子细胞中。我们最近研究了酸性温泉中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）。该质粒稳定遗传的工具包是一个三组分机器，显示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-末端结构域亚基在该平台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组蛋白变体的结构折叠，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-ParB-帕拉多蛋白复合物在DNA上的组装和扩散过程，方法是使用DNase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）和微量热泳（MST）等工具。还将通过使用串联亲和纯化（TAP）方法来检查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。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。