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CRISPR adaptation biases explain preference for acquisition of foreign DNA.

Levy A, Goren MG, Yosef I, Auster O, Manor M, Amitai G, Edgar R, Qimron U, Sorek R - Nature (2015)

Bottom Line: CRISPR-Cas (clustered, regularly interspaced short palindromic repeats coupled with CRISPR-associated proteins) is a bacterial immunity system that protects against invading phages or plasmids.In the process of CRISPR adaptation, short pieces of DNA ('spacers') are acquired from foreign elements and integrated into the CRISPR array.We further show that the avoidance of self is mediated by the RecBCD double-stranded DNA break repair complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.

ABSTRACT
CRISPR-Cas (clustered, regularly interspaced short palindromic repeats coupled with CRISPR-associated proteins) is a bacterial immunity system that protects against invading phages or plasmids. In the process of CRISPR adaptation, short pieces of DNA ('spacers') are acquired from foreign elements and integrated into the CRISPR array. So far, it has remained a mystery how spacers are preferentially acquired from the foreign DNA while the self chromosome is avoided. Here we show that spacer acquisition is replication-dependent, and that DNA breaks formed at stalled replication forks promote spacer acquisition. Chromosomal hotspots of spacer acquisition were confined by Chi sites, which are sequence octamers highly enriched on the bacterial chromosome, suggesting that these sites limit spacer acquisition from self DNA. We further show that the avoidance of self is mediated by the RecBCD double-stranded DNA break repair complex. Our results suggest that, in Escherichia coli, acquisition of new spacers largely depends on RecBCD-mediated processing of double-stranded DNA breaks occurring primarily at replication forks, and that the preference for foreign DNA is achieved through the higher density of Chi sites on the self chromosome, in combination with the higher number of forks on the foreign DNA. This model explains the strong preference to acquire spacers both from high copy plasmids and from phages.

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Related in: MedlinePlus

Dependence of spacer acquisition in replication(A) Spacer acquisition rates in antibiotic-treated E. coli BL21-AI cells. Cells were grown at log phase 16 hours during Cas1+2 induction, with addition of the replication inhibitor nalidixic acid (Nal) or the transcription inhibitor rifampicin (Rif). (B) Spacer acquisition rates of K-12ΔcasCdnaC2 and an isogenic K-12ΔcasC strains during overnight Cas1+2 induction. (C) Spacer acquisition patterns measured following transfer of K-12ΔcasCdnaC2 cells from 39°C to 30°C, during induction of Cas1+2. For all panels, average and error margins for two biological replicates are shown.
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Figure 2: Dependence of spacer acquisition in replication(A) Spacer acquisition rates in antibiotic-treated E. coli BL21-AI cells. Cells were grown at log phase 16 hours during Cas1+2 induction, with addition of the replication inhibitor nalidixic acid (Nal) or the transcription inhibitor rifampicin (Rif). (B) Spacer acquisition rates of K-12ΔcasCdnaC2 and an isogenic K-12ΔcasC strains during overnight Cas1+2 induction. (C) Spacer acquisition patterns measured following transfer of K-12ΔcasCdnaC2 cells from 39°C to 30°C, during induction of Cas1+2. For all panels, average and error margins for two biological replicates are shown.

Mentions: The correlation between spacer acquisition biases and the replication fork stalling sites may suggest that CRISPR adaptation is promoted by active replication of the protospacer-containing DNA. We conducted a series of experiments to test this hypothesis. First, we used the replication-inhibitor quinolone nalidixic acid on E. coli BL21-AI cells during induction of Cas1+2. As a control, we applied the RNA polymerase inhibitor rifampicin that blocks transcription in E. coli but allows DNA replication (this antibiotic does not interfere with transcription of Cas1+2 by the T7 RNA polymerase). Application of nalidixic acid resulted in an almost complete elimination of spacer acquisition (164x-fold reduction), but only ~2-fold reduction in spacer acquisition rates was observed in the rifampicin-treated cells (Fig. 2a; Extended Data Table 1c), providing support to the hypothesis that spacer acquisition depends on DNA replication.


CRISPR adaptation biases explain preference for acquisition of foreign DNA.

Levy A, Goren MG, Yosef I, Auster O, Manor M, Amitai G, Edgar R, Qimron U, Sorek R - Nature (2015)

Dependence of spacer acquisition in replication(A) Spacer acquisition rates in antibiotic-treated E. coli BL21-AI cells. Cells were grown at log phase 16 hours during Cas1+2 induction, with addition of the replication inhibitor nalidixic acid (Nal) or the transcription inhibitor rifampicin (Rif). (B) Spacer acquisition rates of K-12ΔcasCdnaC2 and an isogenic K-12ΔcasC strains during overnight Cas1+2 induction. (C) Spacer acquisition patterns measured following transfer of K-12ΔcasCdnaC2 cells from 39°C to 30°C, during induction of Cas1+2. For all panels, average and error margins for two biological replicates are shown.
© Copyright Policy - permissions-link
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4561520&req=5

Figure 2: Dependence of spacer acquisition in replication(A) Spacer acquisition rates in antibiotic-treated E. coli BL21-AI cells. Cells were grown at log phase 16 hours during Cas1+2 induction, with addition of the replication inhibitor nalidixic acid (Nal) or the transcription inhibitor rifampicin (Rif). (B) Spacer acquisition rates of K-12ΔcasCdnaC2 and an isogenic K-12ΔcasC strains during overnight Cas1+2 induction. (C) Spacer acquisition patterns measured following transfer of K-12ΔcasCdnaC2 cells from 39°C to 30°C, during induction of Cas1+2. For all panels, average and error margins for two biological replicates are shown.
Mentions: The correlation between spacer acquisition biases and the replication fork stalling sites may suggest that CRISPR adaptation is promoted by active replication of the protospacer-containing DNA. We conducted a series of experiments to test this hypothesis. First, we used the replication-inhibitor quinolone nalidixic acid on E. coli BL21-AI cells during induction of Cas1+2. As a control, we applied the RNA polymerase inhibitor rifampicin that blocks transcription in E. coli but allows DNA replication (this antibiotic does not interfere with transcription of Cas1+2 by the T7 RNA polymerase). Application of nalidixic acid resulted in an almost complete elimination of spacer acquisition (164x-fold reduction), but only ~2-fold reduction in spacer acquisition rates was observed in the rifampicin-treated cells (Fig. 2a; Extended Data Table 1c), providing support to the hypothesis that spacer acquisition depends on DNA replication.

Bottom Line: CRISPR-Cas (clustered, regularly interspaced short palindromic repeats coupled with CRISPR-associated proteins) is a bacterial immunity system that protects against invading phages or plasmids.In the process of CRISPR adaptation, short pieces of DNA ('spacers') are acquired from foreign elements and integrated into the CRISPR array.We further show that the avoidance of self is mediated by the RecBCD double-stranded DNA break repair complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.

ABSTRACT
CRISPR-Cas (clustered, regularly interspaced short palindromic repeats coupled with CRISPR-associated proteins) is a bacterial immunity system that protects against invading phages or plasmids. In the process of CRISPR adaptation, short pieces of DNA ('spacers') are acquired from foreign elements and integrated into the CRISPR array. So far, it has remained a mystery how spacers are preferentially acquired from the foreign DNA while the self chromosome is avoided. Here we show that spacer acquisition is replication-dependent, and that DNA breaks formed at stalled replication forks promote spacer acquisition. Chromosomal hotspots of spacer acquisition were confined by Chi sites, which are sequence octamers highly enriched on the bacterial chromosome, suggesting that these sites limit spacer acquisition from self DNA. We further show that the avoidance of self is mediated by the RecBCD double-stranded DNA break repair complex. Our results suggest that, in Escherichia coli, acquisition of new spacers largely depends on RecBCD-mediated processing of double-stranded DNA breaks occurring primarily at replication forks, and that the preference for foreign DNA is achieved through the higher density of Chi sites on the self chromosome, in combination with the higher number of forks on the foreign DNA. This model explains the strong preference to acquire spacers both from high copy plasmids and from phages.

Show MeSH
Related in: MedlinePlus