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

Bhuvan Dixit; Vineet Anand; Md Saddam Hussain; Manish Kumar

doi:10.1016/j.crmicr.2021.100040

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

Curr Res Microb Sci. 2021 May 29:2:100040. doi: 10.1016/j.crmicr.2021.100040. eCollection 2021 Dec.

Authors

Bhuvan Dixit¹, Vineet Anand¹, Md Saddam Hussain¹, Manish Kumar¹

Affiliation

¹ Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, 781039, Assam, India.

Abstract

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 (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.

Keywords: CRISPR-Cas; DNase; Leptospira; nucleolytic.