Target DNA recognition and cleavage by a reconstituted Type I-G CRISPR-Cas immune effector complex

ABSTRACTIn type I CRISPR-Cas system, Cas3 –a nuclease cum helicase– in cooperation with Cascade surveillance complex cleaves the target DNA. Unlike the Cascade/I-E, which is composed of five subunits, the Cascade/I-C is made of only three subunits lacking the CRISPR RNA processing enzyme Cas6, whose role is assumed by Cas5. How these differences in the composition and organisation of Cascade subunits in type I-C influences the Cas3/I-C binding and its target cleavage mechanism is poorly understood. Here, we show that Cas3/I-C is intrinsically a single-strand specific promiscuous nuclease. Apart from the helicase domain, a constellation of highly conserved residues –that are unique to type I-C– located in the uncharacterised C-terminal domain appears to influence the nuclease activity. Recruited by Cascade/I-C, the HD nuclease of Cas3/I-C nicks the single-stranded region of the nontarget strand and positions the helicase motor. Powered by ATP, the helicase motor reels in the target DNA, until it encounters the roadblock en route, which stimulates the HD nuclease. Remarkably, we show that Cas3/I-C supplants Cas3/I-E for CRISPR interference in type I-E in vivo, suggesting that the target cleavage mechanism is evolutionarily conserved between type I-C and type I-E despite the architectural difference exhibited by Cascade/I-C and Cascade/I-E.

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Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3

10.1101/2021.07.18.452824 ◽

2021 ◽

Author(s):

Kazuto Yoshimi ◽

Kohei TAKESHITA ◽

Noriyuki Kodera ◽

Satomi Shibumura ◽

Yuko Yamauchi ◽

...

Keyword(s):

Escherichia Coli ◽

High Speed ◽

Dna Helicase ◽

Type I ◽

Loop Formation ◽

Force Microscopy ◽

Atomic Force ◽

Target Strand ◽

Target Dna

Type I CRISPR-Cas3 uses an RNA-guided multi Cas-protein complex, Cascade, which detects and degrades foreign nucleic acids via the helicase-nuclease Cas3 protein. Despite many studies using cryoEM and smFRET, the precise mechanism of Cas3-mediated cleavage and degradation of target DNA remains elusive. Here we reconstitute the CRISPR-Cas3 system in vitro to show how the Escherichia coli Cas3 (EcoCas3) with EcoCascade exhibits collateral non-specific ssDNA cleavage and target specific DNA degradation. Partial binding of EcoCascade to target DNA with tolerated mismatches within the spacer sequence, but not the PAM, elicits collateral ssDNA cleavage activity of recruited EcoCas3. Conversely, stable binding with complete R-loop formation drives EcoCas3 to nick the non-target strand (NTS) in the bound DNA. Helicase-dependent unwinding then combines with trans ssDNA cleavage of the target strand and repetitive cis cleavage of the NTS to degrade the target dsDNA substrate. High-speed atomic force microscopy demonstrates that EcoCas3 bound to EcoCascade repeatedly reels and releases the target DNA, followed by target fragmentation. Together, these results provide a revised model for collateral ssDNA cleavage and target dsDNA degradation by CRISPR-Cas3, furthering understanding of type I CRISPR priming and interference and informing future genome editing tools.

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DNA Recognition/Processing | DNA Topoisomerases: Type I

Encyclopedia of Biological Chemistry III ◽

10.1016/b978-0-12-819460-7.00557-0 ◽

2013 ◽

pp. 472-478

Author(s):

J.J. Champoux

Keyword(s):

Dna Recognition ◽

Type I ◽

Dna Topoisomerases

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Macroevolution by transposition: drastic modification of DNA recognition by a type I restriction enzyme following Tn5 transposition.

The EMBO Journal ◽

10.1002/j.1460-2075.1993.tb06147.x ◽

1993 ◽

Vol 12 (12) ◽

pp. 4585-4591 ◽

Cited By ~ 37

Author(s):

J. Meister ◽

M. MacWilliams ◽

P. Hübner ◽

H. Jütte ◽

E. Skrzypek ◽

...

Keyword(s):

Restriction Enzyme ◽

Dna Recognition ◽

Type I

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Structural basis for inhibition of the type I-F CRISPR–Cas surveillance complex by AcrIF4, AcrIF7 and AcrIF14

Nucleic Acids Research ◽

10.1093/nar/gkaa1199 ◽

2020 ◽

Author(s):

Clinton Gabel ◽

Zhuang Li ◽

Heng Zhang ◽

Leifu Chang

Keyword(s):

Genome Editing ◽

Conformational Changes ◽

Structural Basis ◽

Type I ◽

Structural Detail ◽

Immune Systems ◽

Helical Bundle ◽

Adaptive Immune ◽

Target Dna ◽

Adaptive Immune Systems

Abstract CRISPR–Cas systems are adaptive immune systems in bacteria and archaea to defend against mobile genetic elements (MGEs) and have been repurposed as genome editing tools. Anti-CRISPR (Acr) proteins are produced by MGEs to counteract CRISPR–Cas systems and can be used to regulate genome editing by CRISPR techniques. Here, we report the cryo-EM structures of three type I-F Acr proteins, AcrIF4, AcrIF7 and AcrIF14, bound to the type I-F CRISPR–Cas surveillance complex (the Csy complex) from Pseudomonas aeruginosa. AcrIF4 binds to an unprecedented site on the C-terminal helical bundle of Cas8f subunit, precluding conformational changes required for activation of the Csy complex. AcrIF7 mimics the PAM duplex of target DNA and is bound to the N-terminal DNA vise of Cas8f. Two copies of AcrIF14 bind to the thumb domains of Cas7.4f and Cas7.6f, preventing hybridization between target DNA and the crRNA. Our results reveal structural detail of three AcrIF proteins, each binding to a different site on the Csy complex for inhibiting degradation of MGEs.

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Structural basis for DNA recognition by the transcription regulator MetR

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x16006828 ◽

2016 ◽

Vol 72 (6) ◽

pp. 417-426 ◽

Cited By ~ 3

Author(s):

Avinash S. Punekar ◽

Jonathan Porter ◽

Stephen B. Carr ◽

Simon E. V. Phillips

Keyword(s):

Molecular Level ◽

Dna Recognition ◽

Data Driven ◽

Structural Basis ◽

Methionine Biosynthesis ◽

Dna Complexes ◽

Winged Helix ◽

Target Dna ◽

Operator Sequence ◽

Dna Complex

MetR, a LysR-type transcriptional regulator (LTTR), has been extensively studied owing to its role in the control of methionine biosynthesis in proteobacteria. A MetR homodimer binds to a 24-base-pair operator region of themetgenes and specifically recognizes the interrupted palindromic sequence 5′-TGAA-N5-TTCA-3′. Mechanistic details underlying the interaction of MetR with its target DNA at the molecular level remain unknown. In this work, the crystal structure of the DNA-binding domain (DBD) of MetR was determined at 2.16 Å resolution. MetR-DBD adopts a winged-helix–turn–helix (wHTH) motif and shares significant fold similarity with the DBD of the LTTR protein BenM. Furthermore, a data-driven macromolecular-docking strategy was used to model the structure of MetR-DBD bound to DNA, which revealed that a bent conformation of DNA is required for the recognition helix α3 and the wing loop of the wHTH motif to interact with the major and minor grooves, respectively. Comparison of the MetR-DBD–DNA complex with the crystal structures of other LTTR-DBD–DNA complexes revealed residues that may confer operator-sequence binding specificity for MetR. Taken together, the results show that MetR-DBD uses a combination of direct base-specific interactions and indirect shape recognition of the promoter to regulate the transcription ofmetgenes.

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