DnaQ exonuclease‐like domain of Cas2 promotes spacer integration in a type I‐E CRISPR‐Cas system

Drabavicius, Gediminas; Šinkūnas, Tomas; Šilanskas, Arūnas; Gasiūnas, Giedrius; Venclovas, C̆eslovas; Šikšnys, Virginijus

doi:10.15252/embr.201745543

Cited by 32 publications

(38 citation statements)

References 45 publications

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“…The type I-E CRISPR system of Streptococcus thermophilus contains a Cas2 that is fused to a DnaQ-like domain. This DnaQ-like domain functions as a 3Ј-5Ј exonuclease that trims prespacer 3Ј overhangs to promote integration (37). In this species, in vitro integration of an unprocessed prespacer by DnaQmutant complexes is undetectable (37), which is consistent with our data ( Fig.…”

Section: Dnaq and Exot Recover Integration Of Unprocessed Prespacerssupporting

confidence: 91%

“…This DnaQ-like domain functions as a 3Ј-5Ј exonuclease that trims prespacer 3Ј overhangs to promote integration (37). In this species, in vitro integration of an unprocessed prespacer by DnaQmutant complexes is undetectable (37), which is consistent with our data ( Fig. 1B) and suggests that Cas1-Cas2 cannot efficiently integrate unprocessed prespacers.…”

Section: Dnaq and Exot Recover Integration Of Unprocessed Prespacerssupporting

confidence: 90%

“…1); nor do we detect any cleavage of these prespacers (Fig. 4), which is consistent with studies of the type I-E system from S. thermophilus (37). The addition of DnaQ or ExoT, but not ExoI, recovers integration of unprocessed prespacers and promotes integration in a functional orientation (Figs.…”

Section: Editors' Pick: Prespacer Processing In E Colisupporting

confidence: 89%

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Processing and integration of functionally oriented prespacers in the Escherichia coli CRISPR system depends on bacterial host exonucleases

Ramachandran

Summerville

Learn

et al. 2020

Journal of Biological Chemistry

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CRISPR-Cas systems provide bacteria with adaptive immunity against viruses. During spacer adaptation, the Cas1-Cas2 complex selects fragments of foreign DNA, called prespacers, and integrates them into CRISPR arrays in an orientation that provides functional immunity. Cas4 is involved in both the trimming of prespacers and the cleavage of protospacer adjacent motif (PAM) in several type I CRISPR-Cas systems, but how the prespacers are processed in systems lacking Cas4, such as the type I-E and I-F systems, is not understood. In Escherichia coli, which has a type I-E system, Cas1-Cas2 preferentially selects prespacers with 3′ overhangs via specific recognition of a PAM, but how these prespacers are integrated in a functional orientation in the absence of Cas4 is not known. Using a biochemical approach with purified proteins, as well as integration, prespacer protection, sequencing, and quantitative PCR assays, we show here that the bacterial 3′–5′ exonucleases DnaQ and ExoT can trim long 3′ overhangs of prespacers and promote integration in the correct orientation. We found that trimming by these exonucleases results in an asymmetric intermediate, because Cas1-Cas2 protects the PAM sequence, which helps to define spacer orientation. Our findings implicate the E. coli host 3′–5′ exonucleases DnaQ and ExoT in spacer adaptation and reveal a mechanism by which spacer orientation is defined in E. coli.

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Section: Dnaq and Exot Recover Integration Of Unprocessed Prespacerssupporting

confidence: 91%

Section: Dnaq and Exot Recover Integration Of Unprocessed Prespacerssupporting

confidence: 90%

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Processing and integration of functionally oriented prespacers in the Escherichia coli CRISPR system depends on bacterial host exonucleases

Ramachandran

Summerville

Learn

et al. 2020

Journal of Biological Chemistry

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“…A recent study on the CRISPR system in Streptococcus thermophilus reported that a Cas2 protein with a DnaQ-like domain exhibits trimming activity on PS precursor DNA, resulting in canonical PS DNA with 5-nt 3'-overhangs (Drabavicius et al, 2018). These fusions of Cas2 and DnaQ-like domains have been reported for several other type I-E systems , hinting that proteins with DnaQ-like domains may naturally be essential for PS maturation.…”

Section: Identification Of Prespacer 3'-overhang Trimming Enzymesmentioning

confidence: 81%

Selective Prespacer Processing Ensures Precise CRISPR-Cas Adaptation

Loeff

Colombo

et al. 2019

Preprint

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CRISPR-Cas immunity protects prokaryotes against foreign genetic elements. CRISPR-Cas uses the highly conserved Cas1-Cas2 complex to establish inheritable memory (spacers). It remains elusive how Cas1-Cas2 acquires spacers from cellular DNA fragments (prespacers) and how it integrates them into the CRISPR array in the correct orientation. By using the high spatiotemporal resolution of single-molecule fluorescence, we reveal that Cas1-Cas2 obtains prespacers in various forms including single-stranded DNA and partial duplexes by selecting them in the DNA-length and PAM-dependent manner. Furthermore, we identify DnaQ exonucleases as enzymes that can mature the Cas1-Cas2-loaded precursor prespacers into an integration-competent size. Cas1-Cas2 protects the PAM sequence from maturation, which results in the production of asymmetrically trimmed prespacers and subsequent spacer integration in the correct orientation. This kinetic coordination in prespacer selection and PAM trimming provides comprehensive understanding of the mechanisms that underlie the integration of functional spacers in the CRISPR array.

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“…The methods described above detect the final result of CRISPR adaptation, spacers integrated into the CRISPR array. New spacers are derived from 'prespacers', short pieces of DNA that are generated inside the cell by various processes and then channeled into CRISPR arrays by Cas1 and Cas2 proteins [24,25,34,[50][51][52][69][70][71][72][73][74][75]. The knowledge about prespacer structure is essential for the understanding of CRISPR adaptation preferences and the ability to control it.…”

Section: Detection Of Spacer Precursors In Vivomentioning

confidence: 99%

Detection of CRISPR adaptation

Shiriaeva

Fedorov

Vyhovskyi

et al. 2020

Biochemical Society Transactions

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Prokaryotic adaptive immunity is built when short DNA fragments called spacers are acquired into CRISPR (clustered regularly interspaced short palindromic repeats) arrays. CRISPR adaptation is a multistep process which comprises selection, generation, and incorporation of prespacers into arrays. Once adapted, spacers provide immunity through the recognition of complementary nucleic acid sequences, channeling them for destruction. To prevent deleterious autoimmunity, CRISPR adaptation must therefore be a highly regulated and infrequent process, at least in the absence of genetic invaders. Over the years, ingenious methods to study CRISPR adaptation have been developed. In this paper, we discuss and compare methods that detect CRISPR adaptation and its intermediates in vivo and propose suppressing PCR as a simple modification of a popular assay to monitor spacer acquisition with increased sensitivity.

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DnaQ exonuclease‐like domain of Cas2 promotes spacer integration in a type I‐E CRISPR‐Cas system

Cited by 32 publications

References 45 publications

Processing and integration of functionally oriented prespacers in the Escherichia coli CRISPR system depends on bacterial host exonucleases

Processing and integration of functionally oriented prespacers in the Escherichia coli CRISPR system depends on bacterial host exonucleases

Selective Prespacer Processing Ensures Precise CRISPR-Cas Adaptation

Detection of CRISPR adaptation

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