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

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

doi:10.1016/j.crmicr.2021.100040

Cited by 11 publications

(6 citation statements)

References 57 publications

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“…While type I-A and I-B Cas4 proteins have been demonstrated to have nonspecific nuclease activities in the absence of Cas1 and Cas2 (Dixit et al, 2021; Lemak et al, 2013, 2014), type I-C Cas4 functions as a sequence specific endonuclease that is only activated in the presence of Cas1 and Cas2 (Lee et al, 2018) ( Figure 1C ). To understand this activation, we examined features of Cas4 that interact with either Cas1 or Cas2.…”

Section: Resultsmentioning

confidence: 99%

“…While Cas4 usually exists as a standalone protein, some systems (type I-G, V-B) encode Cas4/1 fusion proteins, providing evidence of a role for Cas4 in adaptation. In vivo studies have demonstrated that Cas4 is essential for efficient spacer acquisition (Li et al, 2014), ensuring acquisition of spacers with correct PAM sequences (Almendros et al, 2019; Dixit et al, 2021; Kieper et al, 2018, 2021; Zhang et al, 2012) and in the correct orientation (Shiimori et al, 2018). Our previous in vitro studies of the type I-C system from Alkalihalobacillus halodurans (previously Bacillus halodurans ) revealed that Cas4 acts as an endonuclease and cleaves specifically and precisely upstream of PAM sequences within 3′ overhangs of prespacers (Lee et al, 2018, 2019).…”

Section: Introductionmentioning

confidence: 99%

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PAM binding ensures orientational integration during Cas4-Cas1-Cas2 mediated CRISPR adaptation

Dhingra

Suresh

Juneja

et al. 2022

Preprint

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Adaptation in CRISPR-Cas systems immunizes bacteria and archaea against mobile genetic elements. In many DNA-targeting systems, the Cas4-Cas1-Cas2 complex is required for selection and processing of DNA segments containing PAM sequences, prior to integration of these "prespacer" substrates as spacers in the CRISPR array. We determined cryo-EM structures of the Cas4-Cas1-Cas2 adaptation complex from the type I-C system that encodes standalone Cas1 and Cas4 proteins. The structures reveal how Cas4 specifically reads out bases within the PAM sequence and how interactions with both Cas1 and Cas2 activate Cas4 endonuclease activity. The Cas4-PAM interaction ensures tight binding between the adaptation complex and the prespacer, significantly enhancing integration of the non-PAM end into the CRISPR array and ensuring correct spacer orientation. Corroborated with our biochemical results, Cas4-Cas1-Cas2 structures with substrates representing various stages of CRISPR adaptation reveal a temporally resolved mechanism for maturation and integration of functional spacers into the CRISPR array.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

PAM binding ensures orientational integration during Cas4-Cas1-Cas2 mediated CRISPR adaptation

Dhingra

Suresh

Juneja

et al. 2022

Preprint

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“…For studying metallonucleases, substituting metal ions is a common practice to understand their role in the nuclease activity ( Dixit et al, 2016 ; Dixit et al, 2021 ). Mg 2+ ion was substituted with other divalent metal ions (Mn 2+ , Ca 2+ , Ni 2+ , Fe 2+ , Cu 2+ , and Zn 2+ ) to explore their (rLinCas2C and rLinCas2C_Lai) preference for DNase activity.…”

Section: Resultsmentioning

confidence: 99%

Structural and functional characterization of Cas2 of CRISPR-Cas subtype I-C lacking the CRISPR component

Anand

Prabhakaran

Gogoi

et al. 2022

Front. Mol. Biosci.

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The genome of pathogenic Leptospira interrogans serovars (Copenhageni and Lai) are predicted to have CRISPR-Cas of subtypes I-B and I-C. Cas2, one of the core Cas proteins, has a crucial role in adaptive defense against foreign nucleic acids. However, subtype I-C lacks the CRISPR element at its loci essential for RNA-mediated adaptive immunity against foreign nucleic acids. The reason for sustaining the expense of cas genes are unknown in the absence of a CRISPR array. Thus, Cas2C was chosen as a representative Cas protein from two well-studied serovars of Leptospira to address whether it is functional. In this study, the recombinant Cas2C of Leptospira serovars Copenhageni (rLinCas2C, 12 kDa) and Lai (rLinCas2C_Lai, 8.6 kDa) were overexpressed and purified. Due to natural frameshift mutation in the cas2c gene of serovar Lai, rLinCas2C_Lai was overexpressed and purified as a partially translated protein. Nevertheless, the recombinant Cas2C from each serovar exhibited metal-dependent DNase and metal-independent RNase activities. The crystal structure of rLinCas2C obtained at the resolution of 2.60 Å revealed the protein is in apostate conformation and contains N- (1–71 amino acids) and C-terminal (72–90 amino acids) regions, with the former possessing a ferredoxin fold. Substitution of the conserved residues (Tyr7, Asp8, Arg33, and Phe39) with alanine and deletion of Loop L2 resulted in compromised DNase activity. On the other hand, a moderate reduction in RNase activity was evident only in selective rLinCas2C mutants. Overall, in the absence of an array, the observed catalytic activity of Cas2C may be required for biological processes distinct from the CRISPR-Cas-associated function.

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“…A phamily was determined using Phamerator by using “pairwise comparisons to generate gene relationships” ( 10 ). As observed in other phamily members, Iter is predicted to code for a Cas4 family exonuclease (ORF26), which may play a role in nucleotide metabolism or CRISPR activity ( 11 ). Iter has 3′ sticky ends, with an 11-bp overhang.…”

Section: Announcementmentioning

confidence: 99%

Characterization and Genome Sequence of Arthrobacter Phage Iter

Daly

Nesbit

Buchholz

et al. 2022

Microbiol Resour Announc

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

Cited by 11 publications

References 57 publications

PAM binding ensures orientational integration during Cas4-Cas1-Cas2 mediated CRISPR adaptation

PAM binding ensures orientational integration during Cas4-Cas1-Cas2 mediated CRISPR adaptation

Structural and functional characterization of Cas2 of CRISPR-Cas subtype I-C lacking the CRISPR component

Characterization and Genome Sequence of Arthrobacter Phage Iter

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