Periodic Reporting for period 1 - MSOPGDM (Mechanistic studies of prokaryotic genome defense mechanisms)
Periodo di rendicontazione: 2019-04-01 al 2021-03-31
Prokaryotes are constantly threatened by a plethora of viruses (bacteriophages) and other genetic invaders. The evolutionary pressure associated with this host-parasite conflict has resulted in the emergence of numerous defense mechanisms that act on various stages of the bacteriophage life cycle. These defense mechanisms include restriction-modification enzymes and CRISPR-Cas systems, which have been proven to be useful tools for genome engineering. The current project set out to provide a mechanistic basis for immunity of two novel and uncharacterized prokaryotic defense systems. To address this question, we aimed to determine the molecular structures and biochemical activities of key effector proteins that are encoded by these systems. Deciphering these mechanisms will be important for our fundamental understanding of these systems that shape host-invader interactions and may additionally provide new tools to expand the genome engineering toolbox
The current project set out to provide a mechanistic basis for prokaryotic host immunity provided by two novel and uncharacterized prokaryotic defense systems. To address this question, we aimed to determine the molecular structures and biochemical activities of key effector proteins that are encoded by these systems. To this end, we established expression and purification protocols for all of the proteins of these systems and the respective proteins were isolated in sufficient quantities and qualities for structural and biochemical characterization. We succeeded in the determination of high-resolution molecular structures of key effector proteins using both X-ray crystallography and cryo-electron microscopy. Lastly, guided by the high-resolution structures we obtained the first insights into the biochemical activities of the systems. The results obtained within the timeframe of this proposal provide a foundation for future studies that advance our understanding the biological functions and mechanisms of prokaryotic host defense systems.
Despite setbacks due to the ongoing covid-19 pandemic, the goals of the project have been partially reached, as the studies conducted in the course of the project have yielded publishable results, which are currently being prepared for publication. The results will also provide a foundation for future mechanistic and functional studies of prokaryotic defense systems.