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

古细菌是与细菌和真核生物一起生活在地球上的单细胞生物。细菌和古细菌都是原核生物，也就是说，它们的DNA并不局限在一个叫做细胞核的单独的隔间里，而细胞核是真核生物（比如面包酵母、真菌、藻类、动物和人类）的一个决定性标志。古细菌无处不在，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 x 10的28次方古细菌细胞：这是一个巨大的数字。从功能和机制的角度来看，古细菌是细菌和真核生物的镶嵌体。嗜热古菌是一种超级微生物，在80摄氏度或更高的温度下，在温泉、火山、深海喷口中繁衍生息，并表现出一些特性，这使得这些生物对极端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细菌，为这一生命领域带来了新的曙光。尽管在这些生物的解码机制方面取得了进展，但迄今为止，对古细菌染色体分离的基本过程还存在知识差距。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Sulfolobus中剖析这一过程，其基因组编码两种蛋白质，SegA和SegB，它们相互作用形成简单的染色体分离机器。这是广泛存在于古生菌（包括未培养的成员）中的基因组分配系统的原型。因此，它是研究染色体分离的一个很好的模型系统。SegA是一种结合ATP分子和DNA的蛋白质，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基因和一个复制起源的区域，DNA序列对复制至关重要。与这些观察结果一致，显微镜研究显示，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更大的斑块。此外，利用高分辨率显微镜进行的体外研究表明，SegB可以桥接遥远的DNA位点，形成环状结构。这些发现提出了一个假设，即SegB可能是介导染色体组织以准备分离的关键角色。我们最近已经解决了SegA， SegB和各自的DNA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复合体介导染色体进入子细胞的分离和分布的机制，并确定这一过程如何与染色体组织耦合。我们将研究正常和突变细胞的染色体结构，通过使用一种技术能够绘制染色体区域之间的远程接触。具有纯化组分的高分辨率单分子显微镜将探测DNA在SegAB结合和远处位点桥接上的压实。SegAB-DNA复合物内部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“铐住”蛋白质并将混合物置于质谱法（一种能够确定相互作用蛋白质质量的技术）中来鉴定。进一步的目标是通过一种被称为低温电子显微镜的生物物理方法来解决整个SegAB-DNA复合物的结构。来自不同研究的拼图碎片将被结合起来，以产生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.