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

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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.