Structural and mechanistic insight into CRISPR-Cas9 inhibition by anti-CRISPR protein AcrIIC4

Phages, plasmids, and other mobile genetic elements express inhibitors of CRISPR-Cas immune systems, known as anti-CRISPR proteins, to protect themselves from targeted destruction. These anti-CRISPRs have been shown to function through very diverse mechanisms. In this work we investigate the activity of an anti-CRISPR isolated from a prophage in Haemophilus parainfluenzae that blocks CRISPR-Cas9 DNA cleavage activity. We determine the three-dimensional crystal struture of AcrIIC4 and show that it binds to the Cas9 Recognition Domain. This binding does not prevent the Cas9-anti-CRISPR complex from interacting with target DNA but does inhibit DNA cleavage. AcrIIC4 likely acts by blocking the conformational changes that allow the HNH and RuvC endonuclease domains to contact the DNA sites to be nicked.

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Indirect Readout of DNA Controls Filamentation and Activation of a Sequence-Specific Endonuclease

10.1101/585943 ◽

2019 ◽

Author(s):

Smarajit Polley ◽

Dmitry Lyumkis ◽

N. C. Horton

Keyword(s):

Conformational Changes ◽

Dna Cleavage ◽

Metal Ion ◽

Structural Changes ◽

Enzyme Mechanism ◽

Sequence Specificity ◽

Dna Sequence Specificity ◽

Cleavage Activity ◽

Dna Cleavage Activity ◽

Indirect Readout

ABSTRACTFilament or run-on oligomer formation by enzymes is increasingly recognized as an important phenomenon with potentially unique regulatory properties and biological roles. SgrAI is an allosterically regulated type II restriction endonuclease that forms run-on oligomeric (ROO) filaments with enhanced DNA cleavage activity and altered sequence specificity. Here, we present the 3.5 Å cryo-electron microscopy structure of the ROO filament of SgrAI bound to a mimic of cleaved primary site DNA and Mg2+. Large conformational changes stabilize a second metal ion cofactor binding site within the catalytic pocket and facilitate assembling a higher-order enzyme form that is competent for rapid DNA cleavage. The structural changes illuminate the mechanistic origin of hyper-accelerated DNA cleavage activity within the filamentous SgrAI form. An analysis of the protein-DNA interface and the stacking of individual nucleotides reveals how indirect DNA readout within filamentous SgrAI enables recognition of substantially more nucleotide sequences than its low-activity form, thereby expanding DNA sequence specificity. Together, substrate DNA binding, indirect readout, and filamentation simultaneously enhance SgrAI’s catalytic activity and modulate substrate preference. This unusual enzyme mechanism may have evolved to perform the specialized functions of bacterial innate immunity in rapid defense against invading phage DNA without causing damage to the host DNA.

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DNA-cleavage activity of the iron(II) complex with optically active ligands, meta- and para-xylyl-linked N’,N’-dipyridylmethyl-cyclohexane-1,2-diamine

Bioorganic & Medicinal Chemistry Letters ◽

10.1016/j.bmcl.2021.127834 ◽

2021 ◽

Vol 36 ◽

pp. 127834

Author(s):

Koichi Kato ◽

Yoshimi Ichimaru ◽

Yoshinori Okuno ◽

Yoshihiro Yamaguchi ◽

Wanchun Jin ◽

...

Keyword(s):

Dna Cleavage ◽

Optically Active ◽

Cleavage Activity ◽

Dna Cleavage Activity

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A YoeB toxin cleaves both RNA and DNA

Scientific Reports ◽

10.1038/s41598-021-82950-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Julia McGillick ◽

Jessica R. Ames ◽

Tamiko Murphy ◽

Christina R. Bourne

Keyword(s):

Dna Cleavage ◽

Divalent Cations ◽

Nuclease Activity ◽

Dose Dependence ◽

Cleavage Activity ◽

Double Stranded Dna ◽

Dna Cleavage Activity ◽

Evolutionarily Conserved ◽

Toxin Protein ◽

Rna And Dna

AbstractType II toxin-antitoxin systems contain a toxin protein, which mediates diverse interactions within the bacterial cell when it is not bound by its cognate antitoxin protein. These toxins provide a rich source of evolutionarily-conserved tertiary folds that mediate diverse catalytic reactions. These properties make toxins of interest in biotechnology applications, and studies of the catalytic mechanisms continue to provide surprises. In the current work, our studies on a YoeB family toxin from Agrobacterium tumefaciens have revealed a conserved ribosome-independent non-specific nuclease activity. We have quantified the RNA and DNA cleavage activity, revealing they have essentially equivalent dose-dependence while differing in requirements for divalent cations and pH sensitivity. The DNA cleavage activity is as a nickase for any topology of double-stranded DNA, as well as cleaving single-stranded DNA. AtYoeB is able to bind to double-stranded DNA with mid-micromolar affinity. Comparison of the ribosome-dependent and -independent reactions demonstrates an approximate tenfold efficiency imparted by the ribosome. This demonstrates YoeB toxins can act as non-specific nucleases, cleaving both RNA and DNA, in the absence of being bound within the ribosome.

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