Probing the mechanisms that couple genome segregation to chromosome organization in Archaea

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。细菌和古生菌是原核生物，它们的DNA并不局限于一个单独的隔室，称为细胞核，细胞核是真核生物（面包酵母、真菌、藻类、动物和人类等）的标志。珊瑚礁无处不在，构成了生物圈的很大一部分。例如，据报道，仅世界海洋就含有大约1.3 × 10的28个古细菌细胞：这是一个巨大的数字。从功能和机制的角度来看，古生菌是来自细菌和真核生物的镶嵌体。嗜热古生菌是超级微生物，在80摄氏度和更高的温度下在温泉，火山，深海喷口中蓬勃发展，并表现出特性，这使得这些生物体对于生命的基础研究非常有趣。最近的研究提出真核生物起源于古生菌，为古生菌的研究提供了新的思路。尽管在古生菌的染色体解码机制方面取得了很大进展，但到目前为止，对古生菌染色体分离的基本过程仍存在着认识上的空白。基因组分离是每个细胞生命周期的关键阶段：遗传物质被复制，然后分离并分布到两个子细胞中。我们打算在古菌硫化叶菌中剖析这一过程，硫化叶菌的基因组编码两种蛋白质SegA和SegB，它们相互作用形成一个简单的染色体分离机器。这是一个基因组分配系统的原型，广泛分布于古细菌中，包括未培养的成员。SegA是一种蛋白质，它结合一种称为ATP的分子和DNA，没有序列偏好。SegB是一种识别特定DNA序列并以高亲和力结合到这些位点的蛋白质。通过进行全基因组实验，我们已经确定多个SegB DNA基序分散在染色体上。大量的位点聚集在含有segAB基因和复制起点之一的区域中，复制起点是对复制至关重要的DNA序列。与这些观察结果相一致，显微镜研究表明，通过结合染色体上的不同位点，SegB形成多个簇，这些簇在许多细胞中合并成较大的斑块。此外，使用高分辨率显微镜进行的体外研究（可以观察单个DNA分子）表明，SegB可以连接遥远的DNA位点，形成环结构。这些发现提出了一个假设，即SegB可能是介导染色体组织准备分离的关键角色。我们最近解决了SegA，SegB和各自与DNA的复合物的三维结构，这为蛋白质的作用机制提供了快照。本项目旨在发现SegAB复合物介导染色体分离和分配到子细胞中的机制，并确定该过程如何与染色体组织相结合。我们将研究正常和突变细胞的染色体结构，通过使用能够映射染色体区域之间的长距离接触的技术。具有纯化组分的高分辨率单分子显微镜将在SegAB结合和桥接远端位点时探测DNA压实。SegAB-DNA复合物内的相互作用以及与细胞中调节因子的关联将通过不可逆地“手铐”蛋白质并将混合物进行质谱分析来鉴定，这是一种能够确定相互作用蛋白质质量的技术。另一个目标是通过生物物理方法（称为冷冻电子显微镜）解决整个SegAB-DNA复合物的结构。来自不同研究的多块拼图将被结合起来，以生成古细菌染色体分离的整体图像。

Archaea are unicellular organisms that populate our planet together with bacteria and eukaryotes. Bacteria and archaea are prokaryotes i.e., their DNA is not confined into a separate compartment, called nucleus, which is a defining hallmark of eukaryotes (baker yeast, fungi, algae, animals and humans to mention some). Archaea are ubiquitous, constituting a large fraction of the biosphere. For example, it has been reported that the world ocean alone contains approximately 1.3 x 10 to the 28 archaeal cells: this is an enormous number. From a functional and mechanistic standpoint, archaea are a mosaic of tesserae from bacteria and eukaryotes.Thermophilic archaea are super microbes thriving at 80 degrees Celsius and higher temperatures in hot springs, volcanoes, deep sea vents and exhibiting properties, which make these organisms extremely interesting for basic studies on life pushed to extremes. Recent studies have proposed that eukaryotes originated from archaea, casting a novel light on this domain of life.Despite the progress made in decoding mechanisms in these organisms, to date there is a knowledge gap on the fundamental process of chromosome segregation in archaea. Genome segregation is a crucial stage of every cell's life cycle: the genetic material is duplicated, then separated and distributed into two daughter cells. We intend to dissect this process in the archaeon Sulfolobus, whose genome encodes two proteins, SegA and SegB, which interact to form a simple chromosome segregation machine. This is the prototype of a genome partitioning system widespread across archaea, including uncultured members. Thus, it represents an excellent model system to study chromosome segregation.SegA is a protein that binds a molecule, known as ATP, and DNA with no sequence preference. SegB is a protein that recognises specific DNA sequences and binds to to these sites with high affinity. By performing genome-wide experiments, we have established that multiple SegB DNA motifs are scattered across the chromosome. A large number of the sites are clustered in the region harbouring the segAB genes and one of the replication origins, DNA sequences crucial for duplication. Consistent with these observations, microscopy investigations have revealed that by binding the different sites on the chromosome SegB forms multiple clusters, which coalesce into larger patches in numerous cells. Moreover, in vitro studies with high-resolution microscopy, which allows to visualize single DNA molecules, have shown that SegB bridges distant DNA sites, forming loop structures. These findings raise the hypothesis that SegB may be a key player in mediating chromosome organization in preparation for segregation. We have recently solved the three-dimensional structures of SegA, SegB and respective complexes with DNA, which provide snapshots into the mechanism of action of the proteins.This project aims to discover the mechanisms adopted by the SegAB complex to mediate the separation and distribution of chromosomes into daughter cells and to establish how this process is coupled to chromosome organization. We will investigate chromosome structure in normal and mutant cells by using a technique able to map long-range contacts between regions of the chromosome. High-resolution single-molecule microscopy with purified components will probe DNA compaction upon SegAB binding and bridging of distant sites. The interactions within the SegAB-DNA complex and associations with regulators in the cell will be identified by irreversibly 'handcuffing' the proteins and subjecting the mix to mass spectrometry, a technique able to determine the mass of interacting proteins. A further objective is solving the structure of the whole SegAB-DNA complex by a biophysical approach, known as cryo electron microscopy. The multiple pieces of the jigsaw from the different investigations will be combined to generate a holistic picture of chromosome segregation in archaea.