| Like eukaryotic organisms, also prokaryotic cells can be infected by viruses. It has recently been discovered that bacteria and archaea possess a novel type of defense system (CRISPR). Compared to other defense systems, the RNA-mediated CRISPR system is considered unique because viruses are recognized at DNA level and subsequently neutralized by small CRISPR RNAs (immunity), sequence information of harmful viruses is stored in the CRISPR locus on the host chromosome (memory), and acquired immunity is passed on to progeny, and as such protects future generations (inheritance). In the past three years, the applicant's group has established a convenient E. coli CRISPR model system (Brouns, Van der Oost et al. (2008) Science). Analysis of this model system has revealed the key components of the CRISPR interference stage, crucial for virus immunity: a protein complex (Cascade) that processes and binds a small CRISPR RNA (crRNA), as well as a predicted helicase/nuclease (Cas3). Ongoing analyses suggest that crRNA guides the Cascade complex to the complementary viral target DNA (Jore, Brouns, Van der Oost et al., unpublished). In addition, the here proposed project will focus on the role of Cas3 in CRISPR interference. The objectives are a detailed analysis of (i) the Cas3 helicase/nuclease activity, (ii) interaction of Cas3 with the previously characterized crRNA-containing Cascade complex, and (iii) association of the Cas3-Cascade-crRNA supercomplex with target DNA. Apart from improving the molecular understanding of the CRISPR system, the gained insight can be applied in biotechnology for protecting microbial production organisms with a "flu-shot". |
