Biochemical and Structural Characterization of Bacterial Effector Complexes in Viral Defense

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) offer an adaptive immune system that protects bacteria and archaea from nucleic acid invaders through an RNA-mediated nucleic acid cleavage mechanism. Our knowledge of CRISPR nucleic acid cleavage mechanisms is limited to three examples of widely different ribonucleoprotein particles that target either DNA or RNA. CRISPR Type I and II silencing complexes have been well studied and shown to target DNA using via a complex named Cascade and the single protein Cas9 respectively. Type III systems can target both DNA and RNA but the mechanism is still not well understood. The work presented here focuses on the silencing complexes in Type III CRISPR systems and shed some light on the activity of the largest subunit of these complexes. Staphylococcus epidermidis belongs to the Type III-A CRISPR system and has been shown to interfere with invading DNA in vivo. The Type III-A CRISPR system is characterized by the presence of Csm1, a Cas10 family of proteins member, that has a permuted histidine-aspartate (HD) and a nucleotidyl cyclase-like domain, both of which contain sequence features characteristic of nucleases. In chapter 2, we show in vitro that a recombinant S. epidermidis Csm1 cleaves single-stranded DNA exonucleolytically in the 3'-5' direction and in a divalent-metal dependent manner. We further showed that its DNA cleavage activity resides in the GGDD motif of the cyclase-like domain rather than the HD domain. Our data suggest that Csm1 might work in the context of an effector complex to degrade invading DNA. Type III-B Cmr complex from Pyrococcus furiosis has been shown to target RNA. Cmr2 is the largest subunit of the complex, and belongs to the Cas10 family; its domains organization is comparable to Csm1. In chapter 3, we present a structural and functional study of Cmr2 showing that Cmr2 is not the catalytic site of the Cmr complex for the RNA-guided RNA cleavage. However, exciting results of a DNA cleavage activity by Cmr2 suggest a possible dual silencing of DNA and RNA by type III-B systems. Taken in total, the work presented in this dissertation provides insights into the silencing mechanism of the effector complexes in viral defense and highlights the role of their largest subunit, the signature protein Cas10, in type III CRISPR systems.