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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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Related in: MedlinePlus

Cas1 requires Cas2 for robust protospacer integrationa, Schematic of the integration assays using 32P-labeled protospacers (PDB code 4P6I for Cas1–Cas2). b, Integration assays in the presence of increasing protein and 10 mM MnCl2. The titration corresponds to 0, 50, 100 and 200 nM protein. c, Same as b except in the presence of 10 mM MgCl2. The data presented in b and c are representative of at least three replicates.
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Figure 7: Cas1 requires Cas2 for robust protospacer integrationa, Schematic of the integration assays using 32P-labeled protospacers (PDB code 4P6I for Cas1–Cas2). b, Integration assays in the presence of increasing protein and 10 mM MnCl2. The titration corresponds to 0, 50, 100 and 200 nM protein. c, Same as b except in the presence of 10 mM MgCl2. The data presented in b and c are representative of at least three replicates.

Mentions: To test whether the Cas1–Cas2 complex is sufficient to catalyze DNA recombination in vitro, assays were conducted using purified Cas1–Cas2 complex, 33 bp protospacer DNA and an acceptor “target” plasmid consisting of the pUC19 backbone with an inserted CRISPR locus (pCRISPR) (Fig 1a). Co-incubation of these reagents converted the supercoiled plasmid into three main products: relaxed and linear plasmid species, and a fast-migrating species we term Band X (Fig. 1b, c and Extended Data Fig. 1a). Product formation required Cas1, Cas2 and the protospacer DNA (Extended Data Fig. 1b-d), and was consistent with previous divalent metal ion-dependent and sequence-nonspecific in vitro activity requirements of Cas117-19 and Cas220-22. Product DNA migration was not affected by treatment with EDTA, EDTA and phenol-chloroform extraction or Proteinase K in the presence of EDTA and detergent (Extended Data Fig. 1e), indicating that product DNAs are unlikely to be bound to Cas1 and/or Cas2. Consistent with product DNA resulting from covalent integration of protospacer DNA into the plasmid, the relaxed and linear forms of pCRISPR became radiolabeled in reactions containing 32P-labeled protospacer DNA (Fig. 1d and Extended Data Fig. 2). Although Cas1 alone catalyzed a low level of protospacer integration in the presence of Mn2+, the reaction was enhanced significantly by the presence of Cas2 (Extended Data Fig. 2b).


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

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

Cas1 requires Cas2 for robust protospacer integrationa, Schematic of the integration assays using 32P-labeled protospacers (PDB code 4P6I for Cas1–Cas2). b, Integration assays in the presence of increasing protein and 10 mM MnCl2. The titration corresponds to 0, 50, 100 and 200 nM protein. c, Same as b except in the presence of 10 mM MgCl2. The data presented in b and c are representative of at least three replicates.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Cas1 requires Cas2 for robust protospacer integrationa, Schematic of the integration assays using 32P-labeled protospacers (PDB code 4P6I for Cas1–Cas2). b, Integration assays in the presence of increasing protein and 10 mM MnCl2. The titration corresponds to 0, 50, 100 and 200 nM protein. c, Same as b except in the presence of 10 mM MgCl2. The data presented in b and c are representative of at least three replicates.
Mentions: To test whether the Cas1–Cas2 complex is sufficient to catalyze DNA recombination in vitro, assays were conducted using purified Cas1–Cas2 complex, 33 bp protospacer DNA and an acceptor “target” plasmid consisting of the pUC19 backbone with an inserted CRISPR locus (pCRISPR) (Fig 1a). Co-incubation of these reagents converted the supercoiled plasmid into three main products: relaxed and linear plasmid species, and a fast-migrating species we term Band X (Fig. 1b, c and Extended Data Fig. 1a). Product formation required Cas1, Cas2 and the protospacer DNA (Extended Data Fig. 1b-d), and was consistent with previous divalent metal ion-dependent and sequence-nonspecific in vitro activity requirements of Cas117-19 and Cas220-22. Product DNA migration was not affected by treatment with EDTA, EDTA and phenol-chloroform extraction or Proteinase K in the presence of EDTA and detergent (Extended Data Fig. 1e), indicating that product DNAs are unlikely to be bound to Cas1 and/or Cas2. Consistent with product DNA resulting from covalent integration of protospacer DNA into the plasmid, the relaxed and linear forms of pCRISPR became radiolabeled in reactions containing 32P-labeled protospacer DNA (Fig. 1d and Extended Data Fig. 2). Although Cas1 alone catalyzed a low level of protospacer integration in the presence of Mn2+, the reaction was enhanced significantly by the presence of Cas2 (Extended Data Fig. 2b).

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