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

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 80C and higher temperatures
and exhibiting unusual properties, which make these organisms valuable
for the development of novel biotechnological applications, but also
interesting 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 genetic
material 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 eukaryotic
cells. 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-ParBParA
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 Cterminus
CenpA-like domain with proteins potentially involved in posttranscriptional
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.