The CRISPR-associated Cas4 protein from 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 serovar Copenhageni strain Fiocruz L1-130, the gene is functionally transcribed as an active component of the CRISPR-Cas I-B system. Investigation of nuclease activity of Cas4 (LinCas4) of the illustrated divalent-metal cofactor (Mn or Mg) dependent endonuclease activity on the DNA substrate. In agreement, mutation of the selective metal interacting residues (Asp and Glu) curtails the DNA cleavage activity in LinCas4. Computational modeling shows metal-ion interacting residues (Asp and Glu) 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 (LinCas4) or one of the four cysteine residues (LinCas4) 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 condition displayed its coordination with four iron atoms per LinCas4 monomer and was confirmed by the UV and CD spectroscopy studies.