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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

A model explaining the preference for spacer acquisition near TerC as compared to TerA in E. coli BL21-AIThe DNA manipulation at the CRISPR region forms a replication fork stalling site, and leads to extensive spacer acquisition upstream to the CRISPR. While the clockwise fork is stalled at the CRISPR, the counterclockwise fork reaches the Ter region and is stalled at the respective Ter site, TerC, leading to extensive spacer acquisition upstream to TerC. Another factor that can contribute to the observed TerC/TerA bias may be that the clockwise replichore in E. coli (oriC to TerA) is longer than the counter clockwise one (oriC to TerC), leading the forks to stall at TerC more often than at TerA.
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Figure 9: A model explaining the preference for spacer acquisition near TerC as compared to TerA in E. coli BL21-AIThe DNA manipulation at the CRISPR region forms a replication fork stalling site, and leads to extensive spacer acquisition upstream to the CRISPR. While the clockwise fork is stalled at the CRISPR, the counterclockwise fork reaches the Ter region and is stalled at the respective Ter site, TerC, leading to extensive spacer acquisition upstream to TerC. Another factor that can contribute to the observed TerC/TerA bias may be that the clockwise replichore in E. coli (oriC to TerA) is longer than the counter clockwise one (oriC to TerC), leading the forks to stall at TerC more often than at TerA.

Mentions: Another hotspot for spacer acquisition was observed just upstream of the CRISPR I array in the E. coli BL21-AI genome (Fig. 3a). This CRISPR-associated protospacer hotspot clearly depends on the CRISPR activity, because no hotspot was observed near the E. coli BL21-AI CRISPR II array, which lacks a leader sequence and is hence inactive 7 (Fig. 3a). Indeed, in E. coli K-12, where both arrays are known to be active, spacer acquisition assays showed a protospacer peak upstream to each of the two arrays (Fig. 3b). The protospacer peaks at the CRISPR region resembled the peaks seen at the Ter sites, in the sense that they were asymmetric with respect to the replication fork direction, implying that activity at the CRISPR array forms a replication fork stalling site. Presumably the DNA nicking occurring after the leader during insertion of a new spacer 13, stalls the replication fork thus generating a fork-dependent hotspot for spacer acquisition. Frequent stalling of the fork at the CRISPR would mean that the fork coming from the other direction will often be stalled for a longer time at the respective Ter site, TerC, waiting for the fork coming from the CRISPR direction to arrive (Extended Data Fig. 4). This may be one of the factors explaining why the TerC site is a much more pronounced protospacer hotspot than the TerA site (Fig. 1b-c). Another factor that can contribute to the observed TerC/TerA bias may be that the clockwise replichore in E. coli (oriC to TerA) is longer than the counter clockwise one (oriC to TerC), leading the forks to naturally stall at TerC more often than at TerA.


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 spacer acquisition near TerC as compared to TerA in E. coli BL21-AIThe DNA manipulation at the CRISPR region forms a replication fork stalling site, and leads to extensive spacer acquisition upstream to the CRISPR. While the clockwise fork is stalled at the CRISPR, the counterclockwise fork reaches the Ter region and is stalled at the respective Ter site, TerC, leading to extensive spacer acquisition upstream to TerC. Another factor that can contribute to the observed TerC/TerA bias may be that the clockwise replichore in E. coli (oriC to TerA) is longer than the counter clockwise one (oriC to TerC), leading the forks to stall at TerC more often than at TerA.
© Copyright Policy - permissions-link
Related In: Results  -  Collection

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

Figure 9: A model explaining the preference for spacer acquisition near TerC as compared to TerA in E. coli BL21-AIThe DNA manipulation at the CRISPR region forms a replication fork stalling site, and leads to extensive spacer acquisition upstream to the CRISPR. While the clockwise fork is stalled at the CRISPR, the counterclockwise fork reaches the Ter region and is stalled at the respective Ter site, TerC, leading to extensive spacer acquisition upstream to TerC. Another factor that can contribute to the observed TerC/TerA bias may be that the clockwise replichore in E. coli (oriC to TerA) is longer than the counter clockwise one (oriC to TerC), leading the forks to stall at TerC more often than at TerA.
Mentions: Another hotspot for spacer acquisition was observed just upstream of the CRISPR I array in the E. coli BL21-AI genome (Fig. 3a). This CRISPR-associated protospacer hotspot clearly depends on the CRISPR activity, because no hotspot was observed near the E. coli BL21-AI CRISPR II array, which lacks a leader sequence and is hence inactive 7 (Fig. 3a). Indeed, in E. coli K-12, where both arrays are known to be active, spacer acquisition assays showed a protospacer peak upstream to each of the two arrays (Fig. 3b). The protospacer peaks at the CRISPR region resembled the peaks seen at the Ter sites, in the sense that they were asymmetric with respect to the replication fork direction, implying that activity at the CRISPR array forms a replication fork stalling site. Presumably the DNA nicking occurring after the leader during insertion of a new spacer 13, stalls the replication fork thus generating a fork-dependent hotspot for spacer acquisition. Frequent stalling of the fork at the CRISPR would mean that the fork coming from the other direction will often be stalled for a longer time at the respective Ter site, TerC, waiting for the fork coming from the CRISPR direction to arrive (Extended Data Fig. 4). This may be one of the factors explaining why the TerC site is a much more pronounced protospacer hotspot than the TerA site (Fig. 1b-c). Another factor that can contribute to the observed TerC/TerA bias may be that the clockwise replichore in E. coli (oriC to TerA) is longer than the counter clockwise one (oriC to TerC), leading the forks to naturally stall at TerC more often than at TerA.

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