| Abstract [eng] |
Cascade complex together with the signature protein, Cas3, of the type I CRISPR-Cas system confers resistance against the target DNA; however, molecular mechanism of DNA interference stage remained to be elucidated. Therefore, the main objective of this study was to establish the mechanistic details of the DNA interference stage in type I CRISPR-Cas systems. Type I-E CRISPR4-Cas from Streptococcus thermophilus DGCC7710 strain was used as a model system. In this work, we for the first time provide the biochemical characterization of the Cas3 protein that is a hallmark protein for type I systems. We show here that Cas3 combines both an ssDNA nuclease and ATPase/helicase catalytic activities. We have isolated and established the molecular composition of the S. thermophilus Cascade complex, and demonstrated that it binds DNA targets containing promiscuous PAM sequence. Furthermore, we provide the first experimental evidences for the R-loop formation by a single Cascade molecule that revealed the locking step of the stable R-loops. Last but not least, we show Cascade-triggered directional degradation of the DNA target by the Cas3 protein. Summarising our results, we propose detailed mechanism of DNA interference stage for the type I CRISPR-Cas systems. |
