Award details

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

Reference	BB/X006506/1
Principal Investigator / Supervisor	Dr Tung Le
Co-Investigators / Co-Supervisors
Institution	John Innes Centre
Department	Molecular Microbiology
Funding type	Research
Value (£)	434,218
Status	Current
Type	Research Grant
Start date	01/05/2023
End date	30/04/2026
Duration	36 months

Abstract

Chromosome segregation is a fundamental process in all life forms. It requires the concerted action of dedicated proteins and coordination with cellular transactions, such as DNA organization, replication and cell division. The mechanisms that mediate this cell-cycle event in eukaryotes and bacteria are well established. In contrast, chromosome segregation is poorly defined in archaea, the third domain of life. We have investigated a hybrid machine consisting of two interacting proteins, SegA and SegB, that play a key role in archaeal chromosome segregation. SegA is an orthologue of bacterial Walker ParA proteins. SegB is an archaea-specific DNA-binding factor that recognizes palindromic DNA motifs. Our ChIP-seq studies have revealed multiple SegB binding motifs scattered across the chromosome. Consistent with these results, microscopy has shown that SegB forms multiple foci on the chromosome, which then coalesce into large patches. Moreover, atomic force microscopy studies have shown that SegB bridges distant DNA sites, forming loop structures. These unpublished findings support the hypothesis that SegB might mediate chromosome organization prior to and in preparation for segregation. Moreover, recently we have solved the structures of SegA, SegB and respective DNA complexes, which provide snapshots into the mechanism of action of the proteins. The overarching aim of the project is to establish the mechanisms through which the SegAB complex mediates genome segregation and how this process is coupled to chromosome organization. Cross-disciplinary approaches ranging from chromosome conformation capture and structured illumination microscopy to cross-linking mass spectrometry and cryo electron microscopy will provide transformative, multifaceted insights into the mechanism of action of the SegAB complex. The studies will open new perspectives on chromosome biology that will broaden mechanisms and principles established for the other two domains of life.

Summary

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.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

Associated awards:

BB/X00645X/1 Probing the mechanisms that couple genome segregation to chromosome organization in Archaea

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