权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

A novel DNA segregation model system from Archaea revealing bacterial and eukaryotic linkages

古细菌的新型 DNA 分离模型系统揭示了细菌和真核生物的联系

基本信息

批准号：
BB/R006369/1
负责人：
Daniela Barillà
金额：
$ 54.65万
依托单位：
University of York
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2018
资助国家：
英国
起止时间：
2018 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FR006369%2F1
关键词：
novel DNA segregation model system

项目摘要

Archaea evolved as a domain of life billions of years ago, but they are a relatively recent addition to the map of the universal tree of living organisms. Their discovery 40 years ago represented a major milestone. Archaea are unicellular organisms that populate our planet together with bacteria and eukaryotes. Both bacteria and archaea are prokaryotes, i.e. their genetic material is not wrapped by a membrane into a separate compartment, called nucleus, which is instead a hallmark of eukaryotes (baker yeast, fungi, plants, animals and humans to mention some). Archaea are known to be ubiquitous, constituting a considerable fraction of the biosphere. Their ubiquity and abundance make them key players in regulating biogeochemical cycles on Earth. From a functional and mechanistic standpoint, archaea are a mosaic of features from bacteria and eukaryotes, but they are also characterized by unique features like methane production.Heat-loving archaea are super microbes thriving at 80 degrees C and higher temperatures in hot springs, volcanoes, deep sea vents and exhibiting unusual properties, which make these organisms valuable for the development of novel biotechnological applications, but also extremely interesting for studies on life pushed to extremes. Their ability to grow in extreme environments where no other terrestrial organism can survive has also rejuvenated hopes of discovering extraterrestrial life. 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 fundamental 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 DNA is first duplicated, then separated and equally distributed into the two daughter cells. We intend to study this process in a strain of the heat-loving archaeon Sulfolobus isolated from an acidic hot spring in Japan. This microbe contains a large and a small ring of DNA. The large is called chromosome and the small is designated as plasmid. We have recently investigated three proteins that are encoded by the plasmid, pNOB8, and solved their three-dimensional structures. These proteins assemble into a nanomachine that drives duplicated sister plasmids apart, so that each daughter cell receives the same copy number. We intend to introduce changes in the DNA of the host Sulfolobus strain, so that mutations can be introduced in the genes encoding the proteins to show that these factors are essential for the inheritance of the plasmid. One of the proteins responsible for the inheritance of pNOB8 is AspA. It binds strongly to a special site on the plasmid that acts as a docking site and then associates to adjacent regions spreading on the DNA and forming a helix. We want to investigate whether the plamid contains multiple docking sites for AspA and the process through which this protein stretches on the DNA. The other two proteins of the plasmid segregation nanomachine are ParB and ParA. ParB is an adaptor sitting between AspA and ParA in the complex. Adaptors need to be pliable and ParB is indeed flexible: the protein consists of two domains connected by a flexible linker. One of the ParB domains has a bacterial flavour, whereas the other looks like a eukaryotic protein. We suspect that the ParB region that binds ParA is the flexible linker and we want to test this hypothesis. We intend to investigate if other proteins in Sulfolobus associate with the eukaryotic domain of ParB, modifying and regulating it. Finally, we are eager to gain a snapshot of these proteins in Sulfolobus cells. We are going to use microscopes that will provide high-resolution images of the protein complex and will tell us where it localizes relative to the DNA mass of the chromosome. This analysis will allow to identify patterns that will shed light on the life of the pNOB8 DNA segregation nanomachine.

古生物在数十亿年前就进化成了一个生命领域，但它们是生物体宇宙树地图上相对较新的成员。他们40年前的发现是一个重要的里程碑。微生物是单细胞生物，与细菌和真核生物一起居住在我们的星球上。细菌和古细菌都是原核生物，即它们的遗传物质没有被膜包裹到一个单独的隔室中，称为细胞核，这是真核生物的标志（面包酵母，真菌，植物，动物和人类等等）。众所周知，生物圈是无处不在的，构成了生物圈的相当大的一部分。它们的普遍性和丰富性使它们成为调节地球地球化学循环的关键角色。从功能和机制的角度来看，古细菌是细菌和真核生物特征的马赛克，但它们也具有甲烷产生等独特特征。喜热古细菌是超级微生物，在温泉、火山、深海喷口中在80摄氏度和更高的温度下繁衍生息，并表现出不同寻常的特性，这使得这些生物体对于开发新型生物技术应用具有价值，但也非常有趣的研究生命推向极端。它们能够在其他陆地生物无法生存的极端环境中生长，这也重新燃起了发现外星生命的希望。尽管在过去的四十年里，在解码这些生物体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分离的基本过程的信息很少，该主题仍然是一个等待调查的黑匣子。基因组分离是每个细胞生命周期的关键阶段：DNA首先复制，然后分离并平均分配到两个子细胞中。我们打算研究这一过程中的一个菌株的嗜热古菌硫化叶菌分离的酸性温泉在日本。这种微生物含有一个大的和一个小的DNA环。大的称为染色体，小的称为质粒。我们最近研究了由质粒pNOB 8编码的三种蛋白质，并解决了它们的三维结构。这些蛋白质组装成一个纳米机器，驱动复制的姐妹质粒分开，使每个子细胞获得相同的拷贝数。我们打算在宿主硫化叶菌菌株的DNA中引入变化，以便可以在编码蛋白质的基因中引入突变，以表明这些因子对于质粒的遗传是必不可少的。负责pNOB 8遗传的蛋白质之一是AspA。它与质粒上的一个特殊位点强烈结合，该位点作为对接位点，然后与DNA上的相邻区域结合并形成螺旋。我们想研究质粒是否含有AspA的多个对接位点，以及这种蛋白质在DNA上延伸的过程。质粒分离纳米机器的另外两种蛋白质是ParB和ParA。ParB是复合物中位于AspA和帕拉之间的衔接子。衔接子需要是柔韧的，ParB确实是柔性的：蛋白质由两个结构域组成，通过一个柔性接头连接。其中一个ParB结构域具有细菌的味道，而另一个看起来像真核蛋白。我们怀疑结合帕拉的ParB区域是柔性接头，我们想验证这一假设。我们希望研究Sulfolobus中是否有其他蛋白与ParB的真核结构域结合，对其进行修饰和调控，最后，我们渴望获得Sulfolobus细胞中这些蛋白的快照。我们将使用显微镜，它将提供蛋白质复合物的高分辨率图像，并将告诉我们它相对于染色体DNA质量的定位。该分析将允许识别将揭示pNOB8 DNA分离纳米机器的寿命的模式。