Limits...
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.

Show MeSH

Related in: MedlinePlus

The catalytic activity of Cas1 is required for integrationa, Close-up view of the Cas1 active site with the conserved residues shown in stick configurations (PDB 4P6I) b, Integration assays of purified Cas1 active site mutants complexed with wild type Cas2. c, The same as b except using radiolabeled protospacers. The data presented in b and c are representative of at least three replicates.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4359072&req=5

Figure 8: The catalytic activity of Cas1 is required for integrationa, Close-up view of the Cas1 active site with the conserved residues shown in stick configurations (PDB 4P6I) b, Integration assays of purified Cas1 active site mutants complexed with wild type Cas2. c, The same as b except using radiolabeled protospacers. The data presented in b and c are representative of at least three replicates.

Mentions: Bacteria expressing Cas1 active-site mutants, but not active-site mutants of Cas2, are incapable of acquiring new spacers in vivo, demonstrating the catalytic role of Cas1 during spacer acquisition13,14,16. Consistent with these data, Cas1 active site mutants H208A and D221A were defective for protospacer integration in vitro, whereas the Cas2 E9Q active-site mutant supported integration (Fig. 1c, e and Extended Data Fig. 3). The Cas2 C-terminal β6–β7 deletion mutant, which is defective for complex formation with Cas1 and spacer acquisition in vivo, failed to support Cas1-mediated integrase activity (Fig. 1c, e). We conclude that our in vitro assay recapitulates the in vivo functions of Cas1 and Cas2 during spacer acquisition.


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

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

The catalytic activity of Cas1 is required for integrationa, Close-up view of the Cas1 active site with the conserved residues shown in stick configurations (PDB 4P6I) b, Integration assays of purified Cas1 active site mutants complexed with wild type Cas2. c, The same as b except using radiolabeled protospacers. 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 8: The catalytic activity of Cas1 is required for integrationa, Close-up view of the Cas1 active site with the conserved residues shown in stick configurations (PDB 4P6I) b, Integration assays of purified Cas1 active site mutants complexed with wild type Cas2. c, The same as b except using radiolabeled protospacers. The data presented in b and c are representative of at least three replicates.
Mentions: Bacteria expressing Cas1 active-site mutants, but not active-site mutants of Cas2, are incapable of acquiring new spacers in vivo, demonstrating the catalytic role of Cas1 during spacer acquisition13,14,16. Consistent with these data, Cas1 active site mutants H208A and D221A were defective for protospacer integration in vitro, whereas the Cas2 E9Q active-site mutant supported integration (Fig. 1c, e and Extended Data Fig. 3). The Cas2 C-terminal β6–β7 deletion mutant, which is defective for complex formation with Cas1 and spacer acquisition in vivo, failed to support Cas1-mediated integrase activity (Fig. 1c, e). We conclude that our in vitro assay recapitulates the in vivo functions of Cas1 and Cas2 during spacer acquisition.

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