Characterizing and repurposing prokaryotic Argonaute proteins

Prokaryotes have evolved a diverse arsenal of host-defense systems that provide protection against the constant threat of viruses and plasmids. Characterization of these systems has provided interesting insights in fundamental biology of prokaryotes, and has additionally yielded genetic tools that allow for sequence-specific DNA cleavage (e.g. restriction enzymes, CRISPR-Cas enzymes). Despite having the potential to overcome shortcomings of these established genetic tools, certain other host-defense systems remain poorly understood. The proposed project aims to advance our biological and mechanistic understanding of uncharted prokaryotic Argonaute (pAgo) systems.

Eukaryotic Argonaute proteins (eAgos) mediate RNA-guided RNA interference. In contrast, certain pAgos provide host-defense by utilizing DNA guides to target cognate invading DNA. Potentially, pAgos can be used as programmable sequence-specific genetic tools. However, most currently characterized DNA-targeting pAgos originate from thermophilic organisms and rely on high temperatures to target DNA, making them unsuitable for genome editing purposes. Nevertheless, numerous prokaryotes that live at moderate temperatures (20-37 °C) also harbour pAgos. Yet, it is currently unknown how these pAgos efficiently target invading DNA.

Interestingly, specific subclasses of pAgo genes exclusively occur in operons together with genes encoding predicted helicase and/or nuclease domains. This suggests that functionality of these pAgos is intimately linked to helicase and/or nuclease activity. Potentially, these activities facilitate pAgo-DNA targeting by unwinding double-stranded DNA, and/or they could generate overhangs on the targeted DNA. Such activities could respectively enhance pAgo-mediated DNA targeting or promote homologous recombination.

I propose to combine microbiological, biochemical, and X-ray crystallographic techniques to study these mysterious pAgo systems. Detailed understanding of these systems will not only provide fundamental insights into their biological roles and molecular mechanisms, but will additionally give insights in the evolutionary diversification of prokaryotic host-defense systems. Finally, the proposed research will contribute towards the development of novel sequence-specific genetic tools.