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

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

Biography

I did my undergraduate degree at the University of York, completing the 4-year Integrated Masters programme in Biology. I decided that I wished to continue my education by studying for a PhD, and was fortunate to be able to remain in York to do so in the lab of Dr Daniela Barillà. The Barillà group studies the mechanisms underpinning DNA segregation in prokaryotes, using plasmids as model systems. This research project appealed to me primarily due to my interest in the Domain Archaea, a ubiquitous group of organisms that until only four decades ago were classified as bacteria. Archaea often inhabit extreme environments, and so make interesting organisms to study due to their adaptions to these conditions. Understanding fundamental biological processes at the molecular level, here, how DNA is accurately distributed to future cellular generations, is also a interest of mine, and little is known about how this occurs in archaea.

Project summary

The accurate segregation of newly-replicated genetic material to daughter cells is a fundamental process that occurs across the three domains of life. Plasmids, small circular DNA molecules that replicate independently of the main chromosome, are used as models to study genome segregation mechanisms in prokaryotes. Plasmids that are required to be inherited by daughter cells encode genes for their own segregation or partition cassettes, allowing them to be correctly transported to each cell pole prior to division, and ensuring that correct ploidy is maintained in subsequent cellular generations.

I am using the low copy-number plasmid pNOB8, from the hyperthermophilic archaeon Sulfolobus (strain NOB8H2) as a model to investigate archaeal segregation systems. The genetic organisation of the partition cassette has previously been described. Briefly, the partition system comprises three proteins and a specific DNA motif immediately upstream of the cassette. One of the proteins is AspA, a site-specific DNA-binding protein that binds to the motif, and can also spread along the DNA, forming an extended complex. A second DNA sequence motif has previously been identified on plasmid pNOB8.  My project has so far focussed on characterisation of the interactions between the AspA protein and this second binding site. I initially attempted ChIP-Seq (Chromatin Immunoprecipitation and Sequencing) experiments to map AspA:DNA interactions in vivo, however this has proved challenging, particularly as subsequent MinION sequencing of the host Sulfolobus strain genome showed a mosaic of multiple insertions of pNOB8 fragments in the chromosome.

I am currently using in vitro techniques to characterise AspA interactions with the second DNA motif, such as AFM (Atomic Force Microscopy), EMSA (Electromobility-Shift Assay), and a further short term aim is to switch to an in vitro variation of ChIP, IDAP-Seq, to map Asp binding to the pNOB8 plasmid.