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 theyare a relatively recent addition to the universal tree of living organisms.Archaea show a mosaic of tesserae from bacteria and eukaryotes, butthey are also characterized by unique molecular features. Thermophilicarchaea are super microbes thriving at 80C and higher temperaturesand exhibiting unusual properties, which make these organisms valuablefor the development of novel biotechnological applications, but alsointeresting for studies on life pushed to extremes. Thermophilic archaeaare also important for studies on the origin of life and the recentdiscovery of the Lokiarchaeota group has suggested that eukaryotesmight have originated from archaea.Despite the significant progress made in decoding molecularmechanisms in these organisms in the last four decades, to date littleinformation is available on the process of DNA segregation in archaeaand the subject remains a black box awaiting investigation. Genomesegregation is a crucial stage of the life cycle of every cell: the geneticmaterial is first duplicated, then separated and equally distributed intodaughter cells. We have recently investigated the molecular machinery ofthe partition system harboured by a low copy number plasmid in aSulfolobus species from acidic hot springs (Science: 349: 1120-1124). Thetoolkit for the stable inheritance of this plasmid is a three-componentmachine showing linkages to bacterial and eukaryotic proteins. Thissystem encodes a Walker-type ParA, a chimaeric adaptor ParB and acentromere-binding factor, AspA. The AspA protein spreads on the DNAgenerating a helical docking platform onto which ParB N-terminusdomain subunits assemble into a second superhelix. Surprisingly, theParB C-terminus exhibits a structural fold similar to the CenpA histonevariant, which is involved in assembly of the kinetochore in eukaryoticcells. This unique multi-protein structure merges prokaryotic andeukaryotic elements, suggesting the conservation of DNA segregationprinciples across the three domains of life. The project aims toinvestigate the process of assembly and spreading of the AspA-ParBParAmulti-protein complex on the DNA by using tools such as DNasefootprinting and mobility shift assays as well as chromatinimmunoprecipitation (ChIP-Seq), atomic force microscopy (AFM) andmicroscale thermophoresis (MST). The interaction of the ParB CterminusCenpA-like domain with proteins potentially involved in posttranscriptionalmodification of ParB will also be examined by using atandem affinity purification (TAP) approach. An additional objective issequencing the chromosome of the host Sulfolobus strain to establishwhether there is a dynamic flux between chromosome and plasmid.