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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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Distribution of newly acquired spacers on the genome during synchronized replicationE. coli K-12ΔcasCdnaC2 cells were transferred from 39°C (replication restrictive temperature) to 30°C (replication permissive). Cas1+2 were induced in these cells 30 minutes prior to the transfer to 30°C and during the growth in 30°C. At given time points: (A) following 20 minutes; (B) following 40 minutes; (C) following 60 minutes from replication initiation, newly acquired spacers were sequenced. Shown are the positions of the newly acquired spacers in windows of 100kb, and their fraction out of the total new spacers in the sample.
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Figure 8: Distribution of newly acquired spacers on the genome during synchronized replicationE. coli K-12ΔcasCdnaC2 cells were transferred from 39°C (replication restrictive temperature) to 30°C (replication permissive). Cas1+2 were induced in these cells 30 minutes prior to the transfer to 30°C and during the growth in 30°C. At given time points: (A) following 20 minutes; (B) following 40 minutes; (C) following 60 minutes from replication initiation, newly acquired spacers were sequenced. Shown are the positions of the newly acquired spacers in windows of 100kb, and their fraction out of the total new spacers in the sample.

Mentions: We next asked whether spacer acquisition preferences correlate with the position of the replication fork. For this, we transferred a culture of the temperature sensitive dnaC2 cells to 39°C for 70 minutes. Since in this temperature replication re-initiation is inhibited, after 70 minutes there are no more progressing forks in these cells. We then induced Cas1+2 expression for 30 minutes, and transferred the culture to 30°C, resulting in synchronized initiation of replication. At these conditions, it takes the replication forks on average ~60 minutes to complete a full DNA replication cycle in dnaC2 cells 19. In accordance, we sequenced the newly acquired spacers at 20, 40, 60, 90 and 120 minutes following synchronous replication initiation. Strikingly, the fraction of spacers derived from the Ter region has gradually increased with the progression of the replication cycle, reaching 31% after 60 minutes (compared to only 6.4% at the 20 minutes time point; Fig. 2c; Extended Data Fig. 3; Extended Data Table 2b). The pattern repeated itself in the second cycle of replication (90 and 120 minutes; Fig 2c). These results demonstrate temporal correlation between the predicted position of stalled replication forks and the preference to acquire spacers from that position.


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)

Distribution of newly acquired spacers on the genome during synchronized replicationE. coli K-12ΔcasCdnaC2 cells were transferred from 39°C (replication restrictive temperature) to 30°C (replication permissive). Cas1+2 were induced in these cells 30 minutes prior to the transfer to 30°C and during the growth in 30°C. At given time points: (A) following 20 minutes; (B) following 40 minutes; (C) following 60 minutes from replication initiation, newly acquired spacers were sequenced. Shown are the positions of the newly acquired spacers in windows of 100kb, and their fraction out of the total new spacers in the sample.
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Related In: Results  -  Collection

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Figure 8: Distribution of newly acquired spacers on the genome during synchronized replicationE. coli K-12ΔcasCdnaC2 cells were transferred from 39°C (replication restrictive temperature) to 30°C (replication permissive). Cas1+2 were induced in these cells 30 minutes prior to the transfer to 30°C and during the growth in 30°C. At given time points: (A) following 20 minutes; (B) following 40 minutes; (C) following 60 minutes from replication initiation, newly acquired spacers were sequenced. Shown are the positions of the newly acquired spacers in windows of 100kb, and their fraction out of the total new spacers in the sample.
Mentions: We next asked whether spacer acquisition preferences correlate with the position of the replication fork. For this, we transferred a culture of the temperature sensitive dnaC2 cells to 39°C for 70 minutes. Since in this temperature replication re-initiation is inhibited, after 70 minutes there are no more progressing forks in these cells. We then induced Cas1+2 expression for 30 minutes, and transferred the culture to 30°C, resulting in synchronized initiation of replication. At these conditions, it takes the replication forks on average ~60 minutes to complete a full DNA replication cycle in dnaC2 cells 19. In accordance, we sequenced the newly acquired spacers at 20, 40, 60, 90 and 120 minutes following synchronous replication initiation. Strikingly, the fraction of spacers derived from the Ter region has gradually increased with the progression of the replication cycle, reaching 31% after 60 minutes (compared to only 6.4% at the 20 minutes time point; Fig. 2c; Extended Data Fig. 3; Extended Data Table 2b). The pattern repeated itself in the second cycle of replication (90 and 120 minutes; Fig 2c). These results demonstrate temporal correlation between the predicted position of stalled replication forks and the preference to acquire spacers from that position.

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