| Abstract [eng] |
Under constant evolutionary pressure, bacteria developed a set of DNA and RNA-targeting systems to combat their invaders – bacteriophages. Fundamental research on these resistance mechanisms led to the discovery of molecular tools for genetic engineering, such as restriction enzymes, bacterial argonautes and CRISPR-Cas systems. CRISPR-Cas systems are located in the genome part called “defense islands” where genes encoding different antiviral systems are clustered together. The goal of this thesis is to explore the mechanisms of action of type I CRISPR-Cas and CRISPR-Cas associated toxin-antitoxin systems. The first part of this thesis focuses on fundamental aspects of multiprotein class 1 type I-E and I-F CRISPR-Cas effector complexes aiming to understand molecular mechanisms of target recognition and degradation using a combination of biochemical and single molecule assays. The findings presented here show that I E and I F type effectors recognize their dsDNA targets by unidirectional unwinding starting from PAM sequence motif. Furthermore, the complexes can assemble on different length of crRNA and enable dsDNA targeting in a WT-like manner. The second part of the thesis is dedicated to the biochemical and structural characterization of the toxin-antitoxin system HEPN-MNT that is present in the operon of the type I-D CRISPR-Cas system. It is shown that HEPN toxin ribonuclease activity is neutralized by covalent di AMPylation performed by MNT antitoxin. In addition, the data reveals that the unmodified active HEPN toxin cleaves 4 nt from 3′-stem of a range of tRNA, showing a new mechanism of toxin action on tRNA. |
