Characterization of Methanosarcina mazei Solo-Cas4

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) system and its associated (Cas) proteins serve as a defensive mechanism in prokaryotes against foreign genetic elements like viruses, plasmids, or transposons. Thereby, Cas proteins play a key role in CRISPR/Cas systems by recognizing invading nucleic acids and inserting fragments of the foreign nucleic acids (spacer) into the CRISPR array to reminisce overcome future infections. While most Cas proteins are encoded in the CRISPR locus, various cas genes exist that are located outside of CRISPR loci. The function of most of these stand-alone Cas proteins has yet not been investigated. Methanosarcina mazei, an anaerobic methanogenic archaeon, possesses two CRISPR loci together with an additional cas4 gene that has been identified outside of the CRISPR loci encoding a so-called solo-Cas4 protein. The function and structure of this solo-Cas4 protein and the potential connection to the CRISPR/Cas system or a function beyond have not been examined so far. The widespread occurrence of solo-Cas4 proteins even in organisms lacking CRISPR/Cas systems as well as encoded in viral genomes strengthen the hypothesis that solo-Cas4 have a potential function outside the prokaryotic defense.

We heterologously expressed His-tagged SUMO fusion of solo-Cas4 and purified the tag-less protein. The purified solo-Cas4 protein showed a brown coloration and the characteristic absorption spectrum of a 4Fe4S-cluster with absorption peaks at approximately 330 nm and 420 nm. We further showed that M. mazei solo-Cas4 possesses a metal dependent exo- (from 3' to 5') and endo-nuclease activity against ssDNA substrates. The nuclease activity, as well as the 4Fe4S cluster, are characteristic features for Cas4 proteins. However, M. mazei solo-Cas4 showed only low sequence similarity to other Cas4 proteins. The detailed study of M. mazei solo-Cas4 may contribute to a broader understanding and elucidating a distinct role of solo-Cas4 proteins in archaea.