The crystal structure of the CRISPR-associated protein Csn2 from Streptococcus agalactiae

Ellinger, P.; Arslan, Zihni; Wurm, Reinhild; Tschapek, Britta; MacKenzie, Colin R.; Pfeffer, Klaus; Panjikar, Santosh; Wagner, Rolf; Schmitt, Lutz; Gohlke, Holger; Pul, Ümit; Smits, Sander H. J.

doi:10.1016/j.jsb.2012.04.006

Cited by 25 publications

(25 citation statements)

References 62 publications

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“…The similarity with ATPases is limited to the fold core including the region spanning the Walker A and B motifs (75). However, the characteristic amino acids involved in ATP and Mg 2+ binding are replaced and thus Csn2 proteins are not predicted to bind ATP or Mg 2+ , consistent with the experimental results (15,16,19). We also found that previously identified lysine residues involved in DNA binding are not highly conserved even among the short forms of Csn2 (Supplementary Figure S7).…”

Section: Introductionsupporting

confidence: 78%

“…The structurally characterized Csn2 proteins form homotetrameric rings that bind linear double-stranded (ds) DNA through the rings central hole. The major difference between the short and long forms of Csn2 is that the short but not the long forms display Ca 2+ -dependent DNA binding (15,16,19). The specific function of Csn2 in spacer integration remains unclear, but it has been hypothesized that Csn2 is an accessory component that binds the dsDNA ends and protects them from exonucleolytic degradation, while recruiting DNA-repair proteins to seal the breaks (18).…”

Section: Introductionmentioning

confidence: 99%

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Classification and evolution of type II CRISPR-Cas systems

Chylinski

Makarova

Charpentier

et al. 2014

Nucleic Acids Research

415

404

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The CRISPR-Cas systems of archaeal and bacterial adaptive immunity are classified into three types that differ by the repertoires of CRISPR-associated (cas) genes, the organization of cas operons and the structure of repeats in the CRISPR arrays. The simplest among the CRISPR-Cas systems is type II in which the endonuclease activities required for the interference with foreign deoxyribonucleic acid (DNA) are concentrated in a single multidomain protein, Cas9, and are guided by a co-processed dual-tracrRNA:crRNA molecule. This compact enzymatic machinery and readily programmable site-specific DNA targeting make type II systems top candidates for a new generation of powerful tools for genomic engineering. Here we report an updated census of CRISPR-Cas systems in bacterial and archaeal genomes. Type II systems are the rarest, missing in archaea, and represented in ∼5% of bacterial genomes, with an over-representation among pathogens and commensals. Phylogenomic analysis suggests that at least three cas genes, cas1, cas2 and cas4, and the CRISPR repeats of the type II-B system were acquired via recombination with a type I CRISPR-Cas locus. Distant homologs of Cas9 were identified among proteins encoded by diverse transposons, suggesting that type II CRISPR-Cas evolved via recombination of mobile nuclease genes with type I loci.

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Section: Introductionsupporting

confidence: 78%

Section: Introductionmentioning

confidence: 99%

Classification and evolution of type II CRISPR-Cas systems

Chylinski

Makarova

Charpentier

et al. 2014

Nucleic Acids Research

415

404

View full text Add to dashboard Cite

show abstract

“…Since the production of A3A in bacteria or conditions used during the SEC might have led to the observation of artifactual multimers [37], we asked whether such multimers could be observed upon expression of A3A in mammalian cells. We therefore cotransfected 293T cells with expression vectors for HA and FLAG-tagged A3A.…”

Section: Resultsmentioning

confidence: 99%

A DNA Sequence Recognition Loop on APOBEC3A Controls Substrate Specificity

et al. 2014

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APOBEC3A (A3A), one of the seven-member APOBEC3 family of cytidine deaminases, lacks strong antiviral activity against lentiviruses but is a potent inhibitor of adeno-associated virus and endogenous retroelements. In this report, we characterize the biochemical properties of mammalian cell-produced and catalytically active E. coli-produced A3A. The enzyme binds to single-stranded DNA with a Kd of 150 nM and forms dimeric and monomeric fractions. A3A, unlike APOBEC3G (A3G), deaminates DNA substrates nonprocessively. Using a panel of oligonucleotides that contained all possible trinucleotide contexts, we identified the preferred target sequence as TC (A/G). Based on a three-dimensional model of A3A, we identified a putative binding groove that contains residues with the potential to bind substrate DNA and to influence target sequence specificity. Taking advantage of the sequence similarity to the catalytic domain of A3G, we generated A3A/A3G chimeric proteins and analyzed their target site preference. We identified a recognition loop that altered A3A sequence specificity, broadening its target sequence preference. Mutation of amino acids in the predicted DNA binding groove prevented substrate binding, confirming the role of this groove in substrate binding. These findings shed light on how APOBEC3 proteins bind their substrate and determine which sites to deaminate.

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“…In addition to Cas1 and Cas2, some CRISPR–Cas subtypes require the activity of Cas4 or Csn2 to acquire new spacers (1,15,18). Both proteins adopt a toroidal structure (34–36) and are involved in DNA-end metabolism (37,38). Indeed, Cas4 has been shown to form a higher-order protein complex with Cas1/Cas2, termed Cascis ( C RISPR- as sociated c omplex for the i ntegration of s pacers) (39).…”

Section: Introductionmentioning

confidence: 99%

Detection and characterization of spacer integration intermediates in type I-E CRISPR–Cas system

et al. 2014

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The adaptation against foreign nucleic acids by the CRISPR–Cas system (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins) depends on the insertion of foreign nucleic acid-derived sequences into the CRISPR array as novel spacers by still unknown mechanism. We identified and characterized in Escherichia coli intermediate states of spacer integration and mapped the integration site at the chromosomal CRISPR array in vivo. The results show that the insertion of new spacers occurs by site-specific nicking at both strands of the leader proximal repeat in a staggered way and is accompanied by joining of the resulting 5′-ends of the repeat strands with the 3′-ends of the incoming spacer. This concerted cleavage-ligation reaction depends on the metal-binding center of Cas1 protein and requires the presence of Cas2. By acquisition assays using plasmid-located CRISPR array with mutated repeat sequences, we demonstrate that the primary sequence of the first repeat is crucial for cleavage of the CRISPR array and the ligation of new spacer DNA.

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The crystal structure of the CRISPR-associated protein Csn2 from Streptococcus agalactiae

Cited by 25 publications

References 62 publications

Classification and evolution of type II CRISPR-Cas systems

Classification and evolution of type II CRISPR-Cas systems

A DNA Sequence Recognition Loop on APOBEC3A Controls Substrate Specificity

Detection and characterization of spacer integration intermediates in type I-E CRISPR–Cas system

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