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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

Band X corresponds to topoisomers of pCRISPRa, Agarose gel of purified relaxed and Band X integration products. b, Analysis of the total reaction products, after phenol chloroform extraction and ethanol precipitation, on a pre-stained agarose gel. c, Same as b except ethidium bromide staining was performed after electrophoresis. d, PCR amplification products of various segments of pCRISPR using the relaxed, Band X or pCRISPR template shown in a. The laddering effect of minor products using CRISPR locus primers likely reflects the propensity of CRISPR repeats to form DNA hairpins. The data presented in a-d are representative of at least three replicates.
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Figure 9: Band X corresponds to topoisomers of pCRISPRa, Agarose gel of purified relaxed and Band X integration products. b, Analysis of the total reaction products, after phenol chloroform extraction and ethanol precipitation, on a pre-stained agarose gel. c, Same as b except ethidium bromide staining was performed after electrophoresis. d, PCR amplification products of various segments of pCRISPR using the relaxed, Band X or pCRISPR template shown in a. The laddering effect of minor products using CRISPR locus primers likely reflects the propensity of CRISPR repeats to form DNA hairpins. The data presented in a-d are representative of at least three replicates.

Mentions: We observed that Band X did not become radiolabeled in reactions conducted with 32P-labeled protospacer DNA. A time course analysis revealed relaxed DNA product formation within the first minute, followed by accumulation of Band X between 10 and 30 min (Fig. 2d). To determine the properties of Band X, the purified product was analyzed in two different types of agarose gels – one pre-stained with ethidium bromide, similar to the gels presented thus far, and the other stained with ethidium bromide after electrophoresis (post-stained) (Extended Data Fig. 4a). Although Band X migrated as a single species in the pre-stained gel, a ladder of species that migrated faster than the relaxed products was observed in the post-stained gel (Fig. 2e, f). These intermediates are reminiscent of plasmid topoisomers27,28. The same pre- and post-stained agarose gel analysis was performed on the entire integration reaction, generating similar results to those observed with purified Band X (Extended Data Fig. 4b, c). PCR analysis of various segments of pCRISPR using gel-purified Band X as the template yielded amplification products indistinguishable from those generated using unreacted supercoiled pCRISPR or relaxed integration products, supporting the conclusion that Band X corresponds to pCRISPR topoisomers (Extended Data Fig. 4d).


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

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

Band X corresponds to topoisomers of pCRISPRa, Agarose gel of purified relaxed and Band X integration products. b, Analysis of the total reaction products, after phenol chloroform extraction and ethanol precipitation, on a pre-stained agarose gel. c, Same as b except ethidium bromide staining was performed after electrophoresis. d, PCR amplification products of various segments of pCRISPR using the relaxed, Band X or pCRISPR template shown in a. The laddering effect of minor products using CRISPR locus primers likely reflects the propensity of CRISPR repeats to form DNA hairpins. The data presented in a-d 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 9: Band X corresponds to topoisomers of pCRISPRa, Agarose gel of purified relaxed and Band X integration products. b, Analysis of the total reaction products, after phenol chloroform extraction and ethanol precipitation, on a pre-stained agarose gel. c, Same as b except ethidium bromide staining was performed after electrophoresis. d, PCR amplification products of various segments of pCRISPR using the relaxed, Band X or pCRISPR template shown in a. The laddering effect of minor products using CRISPR locus primers likely reflects the propensity of CRISPR repeats to form DNA hairpins. The data presented in a-d are representative of at least three replicates.
Mentions: We observed that Band X did not become radiolabeled in reactions conducted with 32P-labeled protospacer DNA. A time course analysis revealed relaxed DNA product formation within the first minute, followed by accumulation of Band X between 10 and 30 min (Fig. 2d). To determine the properties of Band X, the purified product was analyzed in two different types of agarose gels – one pre-stained with ethidium bromide, similar to the gels presented thus far, and the other stained with ethidium bromide after electrophoresis (post-stained) (Extended Data Fig. 4a). Although Band X migrated as a single species in the pre-stained gel, a ladder of species that migrated faster than the relaxed products was observed in the post-stained gel (Fig. 2e, f). These intermediates are reminiscent of plasmid topoisomers27,28. The same pre- and post-stained agarose gel analysis was performed on the entire integration reaction, generating similar results to those observed with purified Band X (Extended Data Fig. 4b, c). PCR analysis of various segments of pCRISPR using gel-purified Band X as the template yielded amplification products indistinguishable from those generated using unreacted supercoiled pCRISPR or relaxed integration products, supporting the conclusion that Band X corresponds to pCRISPR topoisomers (Extended Data Fig. 4d).

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