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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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Model of protospacer integration during CRISPR–Cas adaptive immunityThe first nucleophilic attack occurs on the minus strand of the first repeat, distal to the leader, by the C 3'-OH end of the protospacer. After half-site intermediate formation, the second integration event occurs on the opposite strand at the leader-repeat border. The resulting single-stranded DNA gaps are repaired by yet uncharacterized mechanisms and the protospacer is fully integrated with the G as the first nucleotide at its 5' end. The asterisk denotes the duplication of the first repeat, as previously observed in vivo13-15.
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Figure 5: Model of protospacer integration during CRISPR–Cas adaptive immunityThe first nucleophilic attack occurs on the minus strand of the first repeat, distal to the leader, by the C 3'-OH end of the protospacer. After half-site intermediate formation, the second integration event occurs on the opposite strand at the leader-repeat border. The resulting single-stranded DNA gaps are repaired by yet uncharacterized mechanisms and the protospacer is fully integrated with the G as the first nucleotide at its 5' end. The asterisk denotes the duplication of the first repeat, as previously observed in vivo13-15.

Mentions: In E. coli, newly acquired spacers harbor a 5' G as the first nucleotide flanking the leader-proximal end of the repeats, which originates from the last nucleotide of the AAG protospacer-adjacent motif (PAM) from foreign DNA13-15,37-39. Such positional specificity is critical for crRNA-guided interference, as a mutation in this position of the corresponding crRNA disrupts PAM binding and subsequent target destruction40-42. We found that ~73% of all integration events into pCRISPR utilized the 3' C end instead of the 3' T end of protospacer DNA during integration (see Fig. 4b for protospacer sequence), and there was a strong preference for this nucleotide to attack the minus strand of the repeat sequence (Fig. 4b,d,e). A similar nucleotide bias was observed in the pUC19 target plasmid sequence data (Fig. 4d). This preference positions the G at the 5' end of the protospacer substrate as the first nucleotide of the newly integrated spacer in the CRISPR locus (Fig. 5). When we used protospacer DNAs lacking a 3' C or bearing 3' C on both ends, the preference for integration into the minus strand of the CRISPR locus was significantly decreased (Extended Data Fig. 9). Thus, the Cas1–Cas2 complex plays a critical role in correctly orienting the C 3'-OH end of protospacer DNA substrates for incorporation within the CRISPR locus.


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

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

Model of protospacer integration during CRISPR–Cas adaptive immunityThe first nucleophilic attack occurs on the minus strand of the first repeat, distal to the leader, by the C 3'-OH end of the protospacer. After half-site intermediate formation, the second integration event occurs on the opposite strand at the leader-repeat border. The resulting single-stranded DNA gaps are repaired by yet uncharacterized mechanisms and the protospacer is fully integrated with the G as the first nucleotide at its 5' end. The asterisk denotes the duplication of the first repeat, as previously observed in vivo13-15.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Model of protospacer integration during CRISPR–Cas adaptive immunityThe first nucleophilic attack occurs on the minus strand of the first repeat, distal to the leader, by the C 3'-OH end of the protospacer. After half-site intermediate formation, the second integration event occurs on the opposite strand at the leader-repeat border. The resulting single-stranded DNA gaps are repaired by yet uncharacterized mechanisms and the protospacer is fully integrated with the G as the first nucleotide at its 5' end. The asterisk denotes the duplication of the first repeat, as previously observed in vivo13-15.
Mentions: In E. coli, newly acquired spacers harbor a 5' G as the first nucleotide flanking the leader-proximal end of the repeats, which originates from the last nucleotide of the AAG protospacer-adjacent motif (PAM) from foreign DNA13-15,37-39. Such positional specificity is critical for crRNA-guided interference, as a mutation in this position of the corresponding crRNA disrupts PAM binding and subsequent target destruction40-42. We found that ~73% of all integration events into pCRISPR utilized the 3' C end instead of the 3' T end of protospacer DNA during integration (see Fig. 4b for protospacer sequence), and there was a strong preference for this nucleotide to attack the minus strand of the repeat sequence (Fig. 4b,d,e). A similar nucleotide bias was observed in the pUC19 target plasmid sequence data (Fig. 4d). This preference positions the G at the 5' end of the protospacer substrate as the first nucleotide of the newly integrated spacer in the CRISPR locus (Fig. 5). When we used protospacer DNAs lacking a 3' C or bearing 3' C on both ends, the preference for integration into the minus strand of the CRISPR locus was significantly decreased (Extended Data Fig. 9). Thus, the Cas1–Cas2 complex plays a critical role in correctly orienting the C 3'-OH end of protospacer DNA substrates for incorporation within the CRISPR locus.

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