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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 catalyzes the disintegration of half-site integrated protospacersa, Schematic of the four strands constituting the Y DNA substrate used in the disintegration assays. b, Native polyacrylamide gel analysis of the annealing products with either Strand A or Strand C radiolabeled. c, Native polyacrylamide gel analysis of disintegration assay products using Y DNA substrates with Strand A labeled. d, Denaturing gel analysis of the disintegration assay products with Strand A labeled.
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Figure 10: Cas1 catalyzes the disintegration of half-site integrated protospacersa, Schematic of the four strands constituting the Y DNA substrate used in the disintegration assays. b, Native polyacrylamide gel analysis of the annealing products with either Strand A or Strand C radiolabeled. c, Native polyacrylamide gel analysis of disintegration assay products using Y DNA substrates with Strand A labeled. d, Denaturing gel analysis of the disintegration assay products with Strand A labeled.

Mentions: We wondered whether Band X arose from protospacer excision from half-site integration products to regenerate pCRISPR in different supercoiled states, analogous to the in vitro disintegration activities of retroviral integrases and transposases (Fig. 2g)29,30. To test this hypothesis, a synthetic Y-structured DNA intermediate that mimics the half-site integration product (Extended Data Fig. 5a,b) was radiolabeled such that the liberated 33 bp protospacer DNA could be detected following disintegration activity. Using this substrate, we observed that Cas1 catalyzed disintegration activity either by itself or in the presence of Cas2 (Fig. 2h). Disintegration activity was confirmed by radiolabeling the 20-nt target DNA strand and monitoring the formation of the joined 40 bp target DNA product (Extended Data Fig. 5c, d). Thus, Cas1–Cas2 integration and disintegration activities are similar to those of retroviral integrases and transposases.


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

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

Cas1 catalyzes the disintegration of half-site integrated protospacersa, Schematic of the four strands constituting the Y DNA substrate used in the disintegration assays. b, Native polyacrylamide gel analysis of the annealing products with either Strand A or Strand C radiolabeled. c, Native polyacrylamide gel analysis of disintegration assay products using Y DNA substrates with Strand A labeled. d, Denaturing gel analysis of the disintegration assay products with Strand A labeled.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 10: Cas1 catalyzes the disintegration of half-site integrated protospacersa, Schematic of the four strands constituting the Y DNA substrate used in the disintegration assays. b, Native polyacrylamide gel analysis of the annealing products with either Strand A or Strand C radiolabeled. c, Native polyacrylamide gel analysis of disintegration assay products using Y DNA substrates with Strand A labeled. d, Denaturing gel analysis of the disintegration assay products with Strand A labeled.
Mentions: We wondered whether Band X arose from protospacer excision from half-site integration products to regenerate pCRISPR in different supercoiled states, analogous to the in vitro disintegration activities of retroviral integrases and transposases (Fig. 2g)29,30. To test this hypothesis, a synthetic Y-structured DNA intermediate that mimics the half-site integration product (Extended Data Fig. 5a,b) was radiolabeled such that the liberated 33 bp protospacer DNA could be detected following disintegration activity. Using this substrate, we observed that Cas1 catalyzed disintegration activity either by itself or in the presence of Cas2 (Fig. 2h). Disintegration activity was confirmed by radiolabeling the 20-nt target DNA strand and monitoring the formation of the joined 40 bp target DNA product (Extended Data Fig. 5c, d). Thus, Cas1–Cas2 integration and disintegration activities are similar to those of retroviral integrases and transposases.

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