The CRISPR-associated Cas4 protein from Leptospira interrogans demonstrate versatile nuclease activity
Bhuvan Dixit, Vineet Anand, Md. Saddam Hussain, Manish Kumar
Abstract
The Cas4 protein is one of the core C RISPR- as sociated (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 (Mn 2+ or Mg 2+ ) dependent endonuclease activity on the DNA substrate. In agreement, mutation of the selective metal interacting residues (Asp 74 and Glu 87 ) curtails the DNA cleavage activity in LinCas4. Computational modeling shows metal-ion interacting residues (Asp 74 and Glu 87 ) 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 Y132A ) or one of the four cysteine residues (LinCas4 C18A ) 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.