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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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Half-site, full-site integration and pCRISPR topoisomer productsa, Schematic of half-site and full-site integration products. b, Linearization of the integration products (lane 4). Lane 3 is the un-treated reaction products. c, Linearization of integration products from radiolabeled protospacer reactions. d, The time course reveals the initial formation of relaxed products, followed by Band X. The inset reveals the products detected using 32P-labeled protospacers. e,f, Analysis of gel-purified relaxed and Band X on agarose gels pre-stained with ethidium bromide (e) or post-stained after electrophoresis (f). g, Schematic of the disintegration reaction. h, Native polyacrylamide gel analysis of the disintegration reaction. The data presented in b-f, h are representative of at least three replicates.
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Figure 2: Half-site, full-site integration and pCRISPR topoisomer productsa, Schematic of half-site and full-site integration products. b, Linearization of the integration products (lane 4). Lane 3 is the un-treated reaction products. c, Linearization of integration products from radiolabeled protospacer reactions. d, The time course reveals the initial formation of relaxed products, followed by Band X. The inset reveals the products detected using 32P-labeled protospacers. e,f, Analysis of gel-purified relaxed and Band X on agarose gels pre-stained with ethidium bromide (e) or post-stained after electrophoresis (f). g, Schematic of the disintegration reaction. h, Native polyacrylamide gel analysis of the disintegration reaction. The data presented in b-f, h are representative of at least three replicates.

Mentions: We tested whether the reaction products of Cas1–Cas2-mediated DNA integration resemble those formed by the strand transfer activity of retroviral integrases and cut-and-paste transposases23-26. These enzymes generate two main products in vitro corresponding to half-site and full-site integration events (Fig. 2a). We observed similar gel mobility of the slowly migrating DNA product generated by Cas1–Cas2 and Nb.BbvCI nickase-digested pCRISPR, consistent with the slow-migrating relaxed DNA species corresponding to half-site products and/or products resulting from full-site integration of one protospacer molecule (Extended Data Fig. 1a). Digestion with EcoRI, which cuts pCRISPR once, converted the reaction products to linear DNAs (Fig. 2b, compare lane 4 to lane 2, and Fig. 2c). We therefore conclude that both the relaxed and Band X DNA products comprise unit-sized pCRISPR circles.


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

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

Half-site, full-site integration and pCRISPR topoisomer productsa, Schematic of half-site and full-site integration products. b, Linearization of the integration products (lane 4). Lane 3 is the un-treated reaction products. c, Linearization of integration products from radiolabeled protospacer reactions. d, The time course reveals the initial formation of relaxed products, followed by Band X. The inset reveals the products detected using 32P-labeled protospacers. e,f, Analysis of gel-purified relaxed and Band X on agarose gels pre-stained with ethidium bromide (e) or post-stained after electrophoresis (f). g, Schematic of the disintegration reaction. h, Native polyacrylamide gel analysis of the disintegration reaction. The data presented in b-f, h are representative of at least three replicates.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Half-site, full-site integration and pCRISPR topoisomer productsa, Schematic of half-site and full-site integration products. b, Linearization of the integration products (lane 4). Lane 3 is the un-treated reaction products. c, Linearization of integration products from radiolabeled protospacer reactions. d, The time course reveals the initial formation of relaxed products, followed by Band X. The inset reveals the products detected using 32P-labeled protospacers. e,f, Analysis of gel-purified relaxed and Band X on agarose gels pre-stained with ethidium bromide (e) or post-stained after electrophoresis (f). g, Schematic of the disintegration reaction. h, Native polyacrylamide gel analysis of the disintegration reaction. The data presented in b-f, h are representative of at least three replicates.
Mentions: We tested whether the reaction products of Cas1–Cas2-mediated DNA integration resemble those formed by the strand transfer activity of retroviral integrases and cut-and-paste transposases23-26. These enzymes generate two main products in vitro corresponding to half-site and full-site integration events (Fig. 2a). We observed similar gel mobility of the slowly migrating DNA product generated by Cas1–Cas2 and Nb.BbvCI nickase-digested pCRISPR, consistent with the slow-migrating relaxed DNA species corresponding to half-site products and/or products resulting from full-site integration of one protospacer molecule (Extended Data Fig. 1a). Digestion with EcoRI, which cuts pCRISPR once, converted the reaction products to linear DNAs (Fig. 2b, compare lane 4 to lane 2, and Fig. 2c). We therefore conclude that both the relaxed and Band X DNA products comprise unit-sized pCRISPR circles.

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