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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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A model explaining the preference for foreign DNA in spacer acquisition(A) RecBCD localizes to double strand DNA breaks (DSBs) and unwinds/degrades the DNA until reaching the nearest properly oriented Chi site. The RecBCD activity generates significant amounts of DNA “debris”, including short and long ssDNA fragments and degraded dsDNA, all of which may serve as substrates for spacer acquisition by Cas1+2. (B) High density of Chi sites on the chromosome reduces spacer acquisition from self DNA. On average, the 8bp-long Chi sites are found every 4.6kb on the E. coli chromosome, 14 times more often than on random DNA. When a DSB occurs on the chromosome, RecBCD DNA degradation activity will quickly be moderated by a nearby Chi site, but a similar DSB on a foreign DNA will lead to much more extensive DNA processing, providing more substrate for spacer acquisition. (C) Preference for spacer acquisition from high copy plasmids. In a replicating cell, most replication forks (blue circles) localize to the multiple copies of the plasmid. Since most DBSs occur during replication 23,26 at stalled replication forks 24,25, plasmid DNA would become more amenable for spacer acquisition. (D) Most phages inject linear DNA into the infected cell. When such linear DNA is not protected, RecBCD will quickly degrade it, providing an intrinsic preference for spacer acquisition from phage DNA.
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Figure 5: A model explaining the preference for foreign DNA in spacer acquisition(A) RecBCD localizes to double strand DNA breaks (DSBs) and unwinds/degrades the DNA until reaching the nearest properly oriented Chi site. The RecBCD activity generates significant amounts of DNA “debris”, including short and long ssDNA fragments and degraded dsDNA, all of which may serve as substrates for spacer acquisition by Cas1+2. (B) High density of Chi sites on the chromosome reduces spacer acquisition from self DNA. On average, the 8bp-long Chi sites are found every 4.6kb on the E. coli chromosome, 14 times more often than on random DNA. When a DSB occurs on the chromosome, RecBCD DNA degradation activity will quickly be moderated by a nearby Chi site, but a similar DSB on a foreign DNA will lead to much more extensive DNA processing, providing more substrate for spacer acquisition. (C) Preference for spacer acquisition from high copy plasmids. In a replicating cell, most replication forks (blue circles) localize to the multiple copies of the plasmid. Since most DBSs occur during replication 23,26 at stalled replication forks 24,25, plasmid DNA would become more amenable for spacer acquisition. (D) Most phages inject linear DNA into the infected cell. When such linear DNA is not protected, RecBCD will quickly degrade it, providing an intrinsic preference for spacer acquisition from phage DNA.

Mentions: The involvement of Chi sites, as points where spacer acquisition activity is terminated, provides another axis for the avoidance of self DNA in CRISPR adaptation. Since the pCas plasmid is completely devoid of Chi sites, its DNA will be fully degraded by RecBCD following any dsDNA break, providing plenty of potential substrate for Cas1+2. In contrast, the high density of Chi sites on the bacterial chromosome serves for the relative avoidance of Cas1+2 to acquire spacers from the chromosome, because RecBCD will only degrade the chromosomal DNA until reaching the nearest Chi site (Fig. 5a-b). Indeed, the ~10 fold higher acquisition frequency from the self choromosome seen in the recB, recC and recD deletion strains conforms with the natural 14-fold enrichment of Chi sites on the chromosome. To further examine whether Chi sites limit spacer acquisition, we performed spacer acquisition experiments with a plasmid that was engineered to contain a cluster of 4 consecutive Chi sites. As expected, an increased preference for chrmomosomal DNA in spacer acquisition was measured for the Chi-containing plasmid (Fig 4g; Extended Data Table 3b; Extended Data Fig. 6).


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)

A model explaining the preference for foreign DNA in spacer acquisition(A) RecBCD localizes to double strand DNA breaks (DSBs) and unwinds/degrades the DNA until reaching the nearest properly oriented Chi site. The RecBCD activity generates significant amounts of DNA “debris”, including short and long ssDNA fragments and degraded dsDNA, all of which may serve as substrates for spacer acquisition by Cas1+2. (B) High density of Chi sites on the chromosome reduces spacer acquisition from self DNA. On average, the 8bp-long Chi sites are found every 4.6kb on the E. coli chromosome, 14 times more often than on random DNA. When a DSB occurs on the chromosome, RecBCD DNA degradation activity will quickly be moderated by a nearby Chi site, but a similar DSB on a foreign DNA will lead to much more extensive DNA processing, providing more substrate for spacer acquisition. (C) Preference for spacer acquisition from high copy plasmids. In a replicating cell, most replication forks (blue circles) localize to the multiple copies of the plasmid. Since most DBSs occur during replication 23,26 at stalled replication forks 24,25, plasmid DNA would become more amenable for spacer acquisition. (D) Most phages inject linear DNA into the infected cell. When such linear DNA is not protected, RecBCD will quickly degrade it, providing an intrinsic preference for spacer acquisition from phage DNA.
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Figure 5: A model explaining the preference for foreign DNA in spacer acquisition(A) RecBCD localizes to double strand DNA breaks (DSBs) and unwinds/degrades the DNA until reaching the nearest properly oriented Chi site. The RecBCD activity generates significant amounts of DNA “debris”, including short and long ssDNA fragments and degraded dsDNA, all of which may serve as substrates for spacer acquisition by Cas1+2. (B) High density of Chi sites on the chromosome reduces spacer acquisition from self DNA. On average, the 8bp-long Chi sites are found every 4.6kb on the E. coli chromosome, 14 times more often than on random DNA. When a DSB occurs on the chromosome, RecBCD DNA degradation activity will quickly be moderated by a nearby Chi site, but a similar DSB on a foreign DNA will lead to much more extensive DNA processing, providing more substrate for spacer acquisition. (C) Preference for spacer acquisition from high copy plasmids. In a replicating cell, most replication forks (blue circles) localize to the multiple copies of the plasmid. Since most DBSs occur during replication 23,26 at stalled replication forks 24,25, plasmid DNA would become more amenable for spacer acquisition. (D) Most phages inject linear DNA into the infected cell. When such linear DNA is not protected, RecBCD will quickly degrade it, providing an intrinsic preference for spacer acquisition from phage DNA.
Mentions: The involvement of Chi sites, as points where spacer acquisition activity is terminated, provides another axis for the avoidance of self DNA in CRISPR adaptation. Since the pCas plasmid is completely devoid of Chi sites, its DNA will be fully degraded by RecBCD following any dsDNA break, providing plenty of potential substrate for Cas1+2. In contrast, the high density of Chi sites on the bacterial chromosome serves for the relative avoidance of Cas1+2 to acquire spacers from the chromosome, because RecBCD will only degrade the chromosomal DNA until reaching the nearest Chi site (Fig. 5a-b). Indeed, the ~10 fold higher acquisition frequency from the self choromosome seen in the recB, recC and recD deletion strains conforms with the natural 14-fold enrichment of Chi sites on the chromosome. To further examine whether Chi sites limit spacer acquisition, we performed spacer acquisition experiments with a plasmid that was engineered to contain a cluster of 4 consecutive Chi sites. As expected, an increased preference for chrmomosomal DNA in spacer acquisition was measured for the Chi-containing plasmid (Fig 4g; Extended Data Table 3b; Extended Data Fig. 6).

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