Evolution of Inosine-Specific Endonuclease V from Bacterial DNase to Eukaryotic RNase

Wu, Jiang‐Lun; Samara, N.L.; Kuraoka, Isao; Yang, Wei

doi:10.1016/j.molcel.2019.06.046

Cited by 37 publications

(57 citation statements)

References 50 publications

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“…Two metal ions separated by a distance of 3.9-4.0 Å were also observed in RNase H1, mouse Endonuclease V, and TtAgo proteins (Fig. 7c) 27,32,34 , suggesting that two metal ions separated by~4 Å is a common feature of the substrate binding state of RuvC and RNase-containing enzymes. Upon the water nucleophile attacking the scissile phosphate, these two metal ions move closer, and as a result, generate a pentacovalent intermediate 32 .…”

Section: Discussionmentioning

confidence: 87%

Structural basis for two metal-ion catalysis of DNA cleavage by Cas12i2

et al. 2020

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To understand how the RuvC catalytic domain of Class 2 Cas proteins cleaves DNA, it will be necessary to elucidate the structures of RuvC-containing Cas complexes in their catalytically competent states. Cas12i2 is a Class 2 type V-I CRISPR-Cas endonuclease that cleaves target dsDNA by an unknown mechanism. Here, we report structures of Cas12i2–crRNA–DNA complexes and a Cas12i2–crRNA complex. We reveal the mechanism of DNA recognition and cleavage by Cas12i2, and activation of the RuvC catalytic pocket induced by a conformational change of the Helical-II domain. The seed region (nucleotides 1–8) is dispensable for RuvC activation, but the duplex of the central spacer (nucleotides 9–15) is required. We captured the catalytic state of Cas12i2, with both metal ions and the ssDNA substrate bound in the RuvC catalytic pocket. Together, our studies provide significant insights into the DNA cleavage mechanism by RuvC-containing Cas proteins.

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Section: Discussionmentioning

confidence: 87%

Structural basis for two metal-ion catalysis of DNA cleavage by Cas12i2

et al. 2020

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“…His8 may interact with Asp193 to facilitate its positioning or the catalytic function. This mechanism is distinct from the two Mg 2+ /Mn 2+ ion-dependent catalytic mechanism employed by EndoV, whose active site consists of a conserved set of carboxylate (Asp and Glu) residues (33,47). However, further studies are needed to fully understand the catalytic mechanism of EndoQ, which could also involve transiently bound metal ion cofactors as observed for mouse EndoV (47).…”

Section: Discussionmentioning

confidence: 90%

Structural basis for recognition of distinct deaminated DNA lesions by endonuclease Q

Shi

Moeller

Banerjee

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Spontaneous deamination of DNA cytosine and adenine into uracil and hypoxanthine, respectively, causes C to T and A to G transition mutations if left unrepaired. Endonuclease Q (EndoQ) initiates the repair of these premutagenic DNA lesions in prokaryotes by cleaving the phosphodiester backbone 5′ of either uracil or hypoxanthine bases or an apurinic/apyrimidinic (AP) lesion generated by the excision of these damaged bases. To understand how EndoQ achieves selectivity toward these structurally diverse substrates without cleaving undamaged DNA, we determined the crystal structures of Pyrococcus furiosus EndoQ bound to DNA substrates containing uracil, hypoxanthine, or an AP lesion. The structures show that substrate engagement by EndoQ depends both on a highly distorted conformation of the DNA backbone, in which the target nucleotide is extruded out of the helix, and direct hydrogen bonds with the deaminated bases. A concerted swing motion of the zinc-binding and C-terminal helical domains of EndoQ toward its catalytic domain allows the enzyme to clamp down on a sharply bent DNA substrate, shaping a deep active-site pocket that accommodates the extruded deaminated base. Within this pocket, uracil and hypoxanthine bases interact with distinct sets of amino acid residues, with positioning mediated by an essential magnesium ion. The EndoQ–DNA complex structures reveal a unique mode of damaged DNA recognition and provide mechanistic insights into the initial step of DNA damage repair by the alternative excision repair pathway. Furthermore, we demonstrate that the unique activity of EndoQ is useful for studying DNA deamination and repair in mammalian systems.

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“…inosine in nucleic acids. [42][43][44] EndoV is present across all domains of life, [45] and naturally utilizes Mg 2+ to cleave inosine-containing substrates.I np rokaryotes,t his activity appears to have evolved for repair of inosine lesions in DNA, [42] while in humans and other higher eukaryotes, EndoV exhibits specific activity toward inosine in RNAa nd is speculated to have roles in degrading A-to-I edited transcripts. [43,44] Interestingly,e nzyme activity can be modulated by replacing Mg 2+ with Ca 2+ ,e nabling EndoV to bind instead of cleave inosine-containing nucleic acid substrates.…”

Section: Introductionmentioning

confidence: 99%

Direct Immunodetection of Global A‐to‐I RNA Editing Activity with a Chemiluminescent Bioassay

et al. 2021

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Adenosine-to-inosine (A-to-I) editing is aconserved eukaryotic RNAmodification that contributes to development, immune response,a nd overall cellular function. Here,w e utilize Endonuclease V( EndoV), whichb inds specifically to inosine in RNA, to develop an EndoV-linked immunosorbency assay(EndoVLISA) as arapid, plate-based chemiluminescent method for measuring global A-to-I editing signatures in cellular RNA. We first optimizea nd validate our assay with chemically synthesized oligonucleotides.W et hen demonstrate rapid detection of inosine content in treated cell lines, demonstrating equivalent performance against current stan-dardRNA-seq approaches.Lastly,wedeploy our EndoVLISA for profiling differential A-to-I RNAe diting signatures in normal and diseased human tissue,illustrating the utility of our platform as ad iagnostic bioassay. Together,t he EndoVLISA method is cost-effective,straightforward, and utilizes common laboratory equipment, offering ah ighly accessible new approach for studying A-to-I editing. Moreover,t he multi-well plate format makes this the first assayamenable for direct highthroughput quantification of A-to-I editing for applications in disease detection and drug development.

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Evolution of Inosine-Specific Endonuclease V from Bacterial DNase to Eukaryotic RNase

Cited by 37 publications

References 50 publications

Structural basis for two metal-ion catalysis of DNA cleavage by Cas12i2

Structural basis for two metal-ion catalysis of DNA cleavage by Cas12i2

Structural basis for recognition of distinct deaminated DNA lesions by endonuclease Q

Direct Immunodetection of Global A‐to‐I RNA Editing Activity with a Chemiluminescent Bioassay

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