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Integrase-mediated spacer acquisition during CRISPR-Cas adaptive immunity.

Nuñez JK, Lee AS, Engelman A, Doudna JA - Nature (2015)

Bottom Line: Here we show that the purified Cas1-Cas2 complex integrates oligonucleotide DNA substrates into acceptor DNA to yield products similar to those generated by retroviral integrases and transposases.Cas1 is the catalytic subunit and Cas2 substantially increases integration activity.Protospacer DNA with free 3'-OH ends and supercoiled target DNA are required, and integration occurs preferentially at the ends of CRISPR repeats and at sequences adjacent to cruciform structures abutting AT-rich regions, similar to the CRISPR leader sequence.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, USA.

ABSTRACT
Bacteria and archaea insert spacer sequences acquired from foreign DNAs into CRISPR loci to generate immunological memory. The Escherichia coli Cas1-Cas2 complex mediates spacer acquisition in vivo, but the molecular mechanism of this process is unknown. Here we show that the purified Cas1-Cas2 complex integrates oligonucleotide DNA substrates into acceptor DNA to yield products similar to those generated by retroviral integrases and transposases. Cas1 is the catalytic subunit and Cas2 substantially increases integration activity. Protospacer DNA with free 3'-OH ends and supercoiled target DNA are required, and integration occurs preferentially at the ends of CRISPR repeats and at sequences adjacent to cruciform structures abutting AT-rich regions, similar to the CRISPR leader sequence. Our results demonstrate the Cas1-Cas2 complex to be the minimal machinery that catalyses spacer DNA acquisition and explain the significance of CRISPR repeats in providing sequence and structural specificity for Cas1-Cas2-mediated adaptive immunity.

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Cas1 tyrosine mutants support integration activity in vitroa, A close-up of the Cas1 active site with the tyrosine residues labeled in blue. b, Structure-based sequence alignment of Cas1 proteins, highlighting the tyrosine residues mutated to alanine in this study. c, Radiolabeled protospacer integration assay of Cas1 tyrosine mutants complexed with WT Cas2. The gel presented in b is representative of at least three replicates.
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Figure 12: Cas1 tyrosine mutants support integration activity in vitroa, A close-up of the Cas1 active site with the tyrosine residues labeled in blue. b, Structure-based sequence alignment of Cas1 proteins, highlighting the tyrosine residues mutated to alanine in this study. c, Radiolabeled protospacer integration assay of Cas1 tyrosine mutants complexed with WT Cas2. The gel presented in b is representative of at least three replicates.

Mentions: Retroviral integration and transposition reactions proceed via nucleophilic attack of DNA 3'-OH groups at target DNA phosphodiester bonds31,32. We found that phosphorylation of both 3'-ends of the protospacer ablated integration, whereas phosphorylation of only one 3' end strongly limited integration (Fig. 3a, b). By analogy to known integrase enzyme mechanisms, DNA integration could proceed by Cas1-catalyzed direct nucleophilic attack of the substrate 3'-OH on the target DNA, or by formation of a Cas1–DNA intermediate, as occurs in the serine and tyrosine families of recombinases33. Four tyrosine residues in the vicinity of the Cas1 active site17-19 could be involved in forming such a covalent intermediate (Extended Data Fig. 7a,b). Purified Cas1 mutant proteins in which each tyrosine was individually changed to alanine each supported protospacer integration in vitro at levels comparable to wild type Cas1–Cas2 (Extended Data Fig. 7c). Thus, the integration reaction likely proceeds via direct nucleophilic attack of protospacer 3'-OH ends onto the target DNA phosphodiester bonds, a mechanism previously hypothesized to occur in vivo34.


Integrase-mediated spacer acquisition during CRISPR-Cas adaptive immunity.

Nuñez JK, Lee AS, Engelman A, Doudna JA - Nature (2015)

Cas1 tyrosine mutants support integration activity in vitroa, A close-up of the Cas1 active site with the tyrosine residues labeled in blue. b, Structure-based sequence alignment of Cas1 proteins, highlighting the tyrosine residues mutated to alanine in this study. c, Radiolabeled protospacer integration assay of Cas1 tyrosine mutants complexed with WT Cas2. The gel presented in b is representative of at least three replicates.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359072&req=5

Figure 12: Cas1 tyrosine mutants support integration activity in vitroa, A close-up of the Cas1 active site with the tyrosine residues labeled in blue. b, Structure-based sequence alignment of Cas1 proteins, highlighting the tyrosine residues mutated to alanine in this study. c, Radiolabeled protospacer integration assay of Cas1 tyrosine mutants complexed with WT Cas2. The gel presented in b is representative of at least three replicates.
Mentions: Retroviral integration and transposition reactions proceed via nucleophilic attack of DNA 3'-OH groups at target DNA phosphodiester bonds31,32. We found that phosphorylation of both 3'-ends of the protospacer ablated integration, whereas phosphorylation of only one 3' end strongly limited integration (Fig. 3a, b). By analogy to known integrase enzyme mechanisms, DNA integration could proceed by Cas1-catalyzed direct nucleophilic attack of the substrate 3'-OH on the target DNA, or by formation of a Cas1–DNA intermediate, as occurs in the serine and tyrosine families of recombinases33. Four tyrosine residues in the vicinity of the Cas1 active site17-19 could be involved in forming such a covalent intermediate (Extended Data Fig. 7a,b). Purified Cas1 mutant proteins in which each tyrosine was individually changed to alanine each supported protospacer integration in vitro at levels comparable to wild type Cas1–Cas2 (Extended Data Fig. 7c). Thus, the integration reaction likely proceeds via direct nucleophilic attack of protospacer 3'-OH ends onto the target DNA phosphodiester bonds, a mechanism previously hypothesized to occur in vivo34.

Bottom Line: Here we show that the purified Cas1-Cas2 complex integrates oligonucleotide DNA substrates into acceptor DNA to yield products similar to those generated by retroviral integrases and transposases.Cas1 is the catalytic subunit and Cas2 substantially increases integration activity.Protospacer DNA with free 3'-OH ends and supercoiled target DNA are required, and integration occurs preferentially at the ends of CRISPR repeats and at sequences adjacent to cruciform structures abutting AT-rich regions, similar to the CRISPR leader sequence.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, USA.

ABSTRACT
Bacteria and archaea insert spacer sequences acquired from foreign DNAs into CRISPR loci to generate immunological memory. The Escherichia coli Cas1-Cas2 complex mediates spacer acquisition in vivo, but the molecular mechanism of this process is unknown. Here we show that the purified Cas1-Cas2 complex integrates oligonucleotide DNA substrates into acceptor DNA to yield products similar to those generated by retroviral integrases and transposases. Cas1 is the catalytic subunit and Cas2 substantially increases integration activity. Protospacer DNA with free 3'-OH ends and supercoiled target DNA are required, and integration occurs preferentially at the ends of CRISPR repeats and at sequences adjacent to cruciform structures abutting AT-rich regions, similar to the CRISPR leader sequence. Our results demonstrate the Cas1-Cas2 complex to be the minimal machinery that catalyses spacer DNA acquisition and explain the significance of CRISPR repeats in providing sequence and structural specificity for Cas1-Cas2-mediated adaptive immunity.

Show MeSH
Related in: MedlinePlus