The CRISPR-associated Cas4 protein from Leptospira interrogans demonstrate versatile nuclease activity.

The Cas4 protein is one of the core CRISPR-associated (Cas) proteins implicated in the adaptation module in many variants of the CRISPR-Cas system in prokaryotes against the invading genetic elements. Cas4 is recognized as a DNA exonuclease that contains a RecB nuclease domain and a Fe-S cluster-binding module. In Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130, the cas4 gene is functionally transcribed as an active component of the CRISPR-Cas I-B system. Investigation of nuclease activity of Cas4 (LinCas4) of the L. interrogans illustrated divalent-metal cofactor (Mn2+ or Mg2+) dependent endonuclease activity on the DNA substrate. In agreement, mutation of the selective metal interacting residues (Asp74 and Glu87) curtails the DNA cleavage activity in LinCas4. Computational modeling shows metal-ion interacting residues (Asp74 and Glu87) in the LinCas4 to be a part of the RecB motifs II and III, the same as other Cas4 orthologs. The mutation of a potential DNA interacting residue in the LinCas4 (LinCas4Y132A) or one of the four cysteine residues (LinCas4C18A) involved in coordinating the 4Fe-4S cluster did not perturb its DNase activity. Iron chelation assay of the purified LinCas4 demonstrated it in the apostate conformation. Reconstitution of the Fe-S cluster in the LinCas4 under in vitro condition displayed its coordination with four iron atoms per LinCas4 monomer and was confirmed by the UV and CD spectroscopy studies.

The core Cas1 protein of CRISPR-Cas I-B in Leptospira shows metal-tunable nuclease activity.

Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 is the causative agent of leptospirosis in animals and humans. This organism carries a functional cas1 gene classified under CRISPR-Cas I-B. In this study, using various nuclease assays and bioinformatics analysis, we report that the recombinant Cas1 (LinCas1) possesses metal-ion dependent DNase activity, which is inhibited upon substitution or chelation of metal-ion and/or interaction with recombinant Cas2 (LinCas2) of L. interrogans. Model of LinCas1 structure shows a shorter N-terminal domain unlike other Cas1 orthologs reported to date. The C-terminal domain of LinCas1 contains conserved divalent-metal binding residues (Glu108, His176, and Glu191) and the mutation of these residues leads to abolition in DNase activity. Immunoassay using anti-LinCas2 demonstrates that LinCas1 interacts with LinCas2 and attains a saturation point. Moreover, the nuclease activity of the LinCas1-Cas2 mixture on ds-DNA displayed a reduction in activity compared to the pure core LinCas proteins under in vitro condition. The DNase activity for LinCas1 is consistent with a role for this protein in the recognition/cleavage of foreign DNA and integration of foreign DNA as spacer into the CRISPR array.

Dual nuclease activity of a Cas2 protein in CRISPR-Cas subtype I-B of Leptospira interrogans.

Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 carries a set of cas genes associated with CRISPR-Cas subtype I-B. Herein, we report for the first time active transcription of a set of cas genes (cas1 to cas8) of L. interrogans where cas4, cas1, cas2 and cas6, cas3, cas8, cas7, cas5 are clustered together in two independent operons. As an initial step toward comprehensive understanding of CRISPR-Cas system in spirochete, the biochemical study of one of the core Leptospira Cas2 proteins (Lep_Cas2) showed nuclease activity on both DNA and RNA in a nonspecific manner. Additionally, unlike other known Cas2 proteins, Lep_Cas2 showed metal-independent RNase activity and preferential activity on RNA over DNA. These results provide insight for understanding Cas2 diversity existing in the prokaryotic adaptive immune system.