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Recovery of arrested replication forks by homologous recombination is error-prone.

Iraqui I, Chekkal Y, Jmari N, Pietrobon V, Fréon K, Costes A, Lambert SA - PLoS Genet. (2012)

Bottom Line: The mutations caused are small insertions/duplications between short tandem repeats (micro-homology) indicative of replication slippage.Our data establish that collapsed forks, but not stalled forks, recovered by homologous recombination are prone to replication slippage.We propose that deletions/insertions, mediated by micro-homology, leading to copy number variations during replication stress may arise by progression of error-prone replication forks restarted by homologous recombination.

View Article: PubMed Central - PubMed

Affiliation: Institut Curie, Centre de Recherche, Orsay, France.

ABSTRACT
Homologous recombination is a universal mechanism that allows repair of DNA and provides support for DNA replication. Homologous recombination is therefore a major pathway that suppresses non-homology-mediated genome instability. Here, we report that recovery of impeded replication forks by homologous recombination is error-prone. Using a fork-arrest-based assay in fission yeast, we demonstrate that a single collapsed fork can cause mutations and large-scale genomic changes, including deletions and translocations. Fork-arrest-induced gross chromosomal rearrangements are mediated by inappropriate ectopic recombination events at the site of collapsed forks. Inverted repeats near the site of fork collapse stimulate large-scale genomic changes up to 1,500 times over spontaneous events. We also show that the high accuracy of DNA replication during S-phase is impaired by impediments to fork progression, since fork-arrest-induced mutation is due to erroneous DNA synthesis during recovery of replication forks. The mutations caused are small insertions/duplications between short tandem repeats (micro-homology) indicative of replication slippage. Our data establish that collapsed forks, but not stalled forks, recovered by homologous recombination are prone to replication slippage. The inaccuracy of DNA synthesis does not rely on PCNA ubiquitination or trans-lesion-synthesis DNA polymerases, and it is not counteracted by mismatch repair. We propose that deletions/insertions, mediated by micro-homology, leading to copy number variations during replication stress may arise by progression of error-prone replication forks restarted by homologous recombination.

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A single fork-arrest induces GCRs that are stimulated by inverted repeats near the site of fork arrest.A. Diagrams of chromosome II containing or not the RTS1 sequence (blue arrow or RTS1-d) and of chromosome III containing ura4+ alone or associated with RTS1-RFB constructs. The RTS1 sequence maps near the mat1 locus where it helps to ensure unidirectional replication [62]. Primers used for amplifying the 1 Kb ura4 fragment or the 650 bp rng3 fragment are depicted in red and grey, respectively. Primers used to amplify the translocation junction (1.2 kb) are represented in orange on chromosome II (TLII) and in black on chromosome III (TLIII). B. Representative PCR-amplifications from 5-FOAR colonies of the indicated strains; ON and OFF refers to the RTS1-RFB being active or not, respectively. PCR products and their sizes are indicated on the figure. C. Effect of intra- and inter-chromosomal recombination between RTS1 repeats on fork-arrest-induced genomic deletion. RTS1-RFB activity and ura4 location with respect to the RFB are given for each construct. The % of deletion events, as determined by the PCR assay, was used to balance the rate of ura4 loss. Then, the RFB-induced deletion rate was calculated by subtracting the rate obtained in the presence of thiamine (Rtf1 being repressed) from the rate obtained in the absence of thiamine (Rtf1 being expressed). The values reported are means of at least 3 independent median rates. Error bars correspond to the standard error (SE). D. Effect of Rqh1 on RFB-induced deletions (left) and translocations (right), as described for panel C. Error bars indicate SE. Statistically significant fold differences between the rqh1-d and the wild-type strains are indicated with an *. E. Representative PCR amplifications from 5-FOAR colonies of the rqh1-d t-ura4<ori strain, as described for panel B. (Refer to Figure S1 for corresponding rates of deletion and translocation when Rtf1 is expressed or not).
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pgen-1002976-g002: A single fork-arrest induces GCRs that are stimulated by inverted repeats near the site of fork arrest.A. Diagrams of chromosome II containing or not the RTS1 sequence (blue arrow or RTS1-d) and of chromosome III containing ura4+ alone or associated with RTS1-RFB constructs. The RTS1 sequence maps near the mat1 locus where it helps to ensure unidirectional replication [62]. Primers used for amplifying the 1 Kb ura4 fragment or the 650 bp rng3 fragment are depicted in red and grey, respectively. Primers used to amplify the translocation junction (1.2 kb) are represented in orange on chromosome II (TLII) and in black on chromosome III (TLIII). B. Representative PCR-amplifications from 5-FOAR colonies of the indicated strains; ON and OFF refers to the RTS1-RFB being active or not, respectively. PCR products and their sizes are indicated on the figure. C. Effect of intra- and inter-chromosomal recombination between RTS1 repeats on fork-arrest-induced genomic deletion. RTS1-RFB activity and ura4 location with respect to the RFB are given for each construct. The % of deletion events, as determined by the PCR assay, was used to balance the rate of ura4 loss. Then, the RFB-induced deletion rate was calculated by subtracting the rate obtained in the presence of thiamine (Rtf1 being repressed) from the rate obtained in the absence of thiamine (Rtf1 being expressed). The values reported are means of at least 3 independent median rates. Error bars correspond to the standard error (SE). D. Effect of Rqh1 on RFB-induced deletions (left) and translocations (right), as described for panel C. Error bars indicate SE. Statistically significant fold differences between the rqh1-d and the wild-type strains are indicated with an *. E. Representative PCR amplifications from 5-FOAR colonies of the rqh1-d t-ura4<ori strain, as described for panel B. (Refer to Figure S1 for corresponding rates of deletion and translocation when Rtf1 is expressed or not).

Mentions: We investigated fork-arrest-induced genome instability by selecting for cell resistance to 5-FOAR, the result of loss of ura4+ function. Inducing fork-arrest at t-ura4<ori increased ura4 loss 3 fold (Table 1). Rtf1 expression in the t-ura4-ori and t>ura4-ori strains did not cause site-specific fork-arrest at ura4 as assessed by 2DGE and did not increase the rate of ura4 loss. Thus, ura4 loss results from the RTS1-RFB activity and not simply from the presence of RTS1 and/or Rtf1 expression (Table 1). To investigate the nature of this genetic instability, primers were designed to amplify the ura4 coding sequence and, as a control, the essential rng3 gene, mapping 30 kb tel-proximal to ura4, that should not be rearranged (Figure 2A and 2B) [35]. The absence of ura4 amplification was classified as a deletion event; sequencing of amplified ura4 sequence was used to identify point mutation events (Figure 2B).


Recovery of arrested replication forks by homologous recombination is error-prone.

Iraqui I, Chekkal Y, Jmari N, Pietrobon V, Fréon K, Costes A, Lambert SA - PLoS Genet. (2012)

A single fork-arrest induces GCRs that are stimulated by inverted repeats near the site of fork arrest.A. Diagrams of chromosome II containing or not the RTS1 sequence (blue arrow or RTS1-d) and of chromosome III containing ura4+ alone or associated with RTS1-RFB constructs. The RTS1 sequence maps near the mat1 locus where it helps to ensure unidirectional replication [62]. Primers used for amplifying the 1 Kb ura4 fragment or the 650 bp rng3 fragment are depicted in red and grey, respectively. Primers used to amplify the translocation junction (1.2 kb) are represented in orange on chromosome II (TLII) and in black on chromosome III (TLIII). B. Representative PCR-amplifications from 5-FOAR colonies of the indicated strains; ON and OFF refers to the RTS1-RFB being active or not, respectively. PCR products and their sizes are indicated on the figure. C. Effect of intra- and inter-chromosomal recombination between RTS1 repeats on fork-arrest-induced genomic deletion. RTS1-RFB activity and ura4 location with respect to the RFB are given for each construct. The % of deletion events, as determined by the PCR assay, was used to balance the rate of ura4 loss. Then, the RFB-induced deletion rate was calculated by subtracting the rate obtained in the presence of thiamine (Rtf1 being repressed) from the rate obtained in the absence of thiamine (Rtf1 being expressed). The values reported are means of at least 3 independent median rates. Error bars correspond to the standard error (SE). D. Effect of Rqh1 on RFB-induced deletions (left) and translocations (right), as described for panel C. Error bars indicate SE. Statistically significant fold differences between the rqh1-d and the wild-type strains are indicated with an *. E. Representative PCR amplifications from 5-FOAR colonies of the rqh1-d t-ura4<ori strain, as described for panel B. (Refer to Figure S1 for corresponding rates of deletion and translocation when Rtf1 is expressed or not).
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getmorefigures.php?uid=PMC3475662&req=5

pgen-1002976-g002: A single fork-arrest induces GCRs that are stimulated by inverted repeats near the site of fork arrest.A. Diagrams of chromosome II containing or not the RTS1 sequence (blue arrow or RTS1-d) and of chromosome III containing ura4+ alone or associated with RTS1-RFB constructs. The RTS1 sequence maps near the mat1 locus where it helps to ensure unidirectional replication [62]. Primers used for amplifying the 1 Kb ura4 fragment or the 650 bp rng3 fragment are depicted in red and grey, respectively. Primers used to amplify the translocation junction (1.2 kb) are represented in orange on chromosome II (TLII) and in black on chromosome III (TLIII). B. Representative PCR-amplifications from 5-FOAR colonies of the indicated strains; ON and OFF refers to the RTS1-RFB being active or not, respectively. PCR products and their sizes are indicated on the figure. C. Effect of intra- and inter-chromosomal recombination between RTS1 repeats on fork-arrest-induced genomic deletion. RTS1-RFB activity and ura4 location with respect to the RFB are given for each construct. The % of deletion events, as determined by the PCR assay, was used to balance the rate of ura4 loss. Then, the RFB-induced deletion rate was calculated by subtracting the rate obtained in the presence of thiamine (Rtf1 being repressed) from the rate obtained in the absence of thiamine (Rtf1 being expressed). The values reported are means of at least 3 independent median rates. Error bars correspond to the standard error (SE). D. Effect of Rqh1 on RFB-induced deletions (left) and translocations (right), as described for panel C. Error bars indicate SE. Statistically significant fold differences between the rqh1-d and the wild-type strains are indicated with an *. E. Representative PCR amplifications from 5-FOAR colonies of the rqh1-d t-ura4<ori strain, as described for panel B. (Refer to Figure S1 for corresponding rates of deletion and translocation when Rtf1 is expressed or not).
Mentions: We investigated fork-arrest-induced genome instability by selecting for cell resistance to 5-FOAR, the result of loss of ura4+ function. Inducing fork-arrest at t-ura4<ori increased ura4 loss 3 fold (Table 1). Rtf1 expression in the t-ura4-ori and t>ura4-ori strains did not cause site-specific fork-arrest at ura4 as assessed by 2DGE and did not increase the rate of ura4 loss. Thus, ura4 loss results from the RTS1-RFB activity and not simply from the presence of RTS1 and/or Rtf1 expression (Table 1). To investigate the nature of this genetic instability, primers were designed to amplify the ura4 coding sequence and, as a control, the essential rng3 gene, mapping 30 kb tel-proximal to ura4, that should not be rearranged (Figure 2A and 2B) [35]. The absence of ura4 amplification was classified as a deletion event; sequencing of amplified ura4 sequence was used to identify point mutation events (Figure 2B).

Bottom Line: The mutations caused are small insertions/duplications between short tandem repeats (micro-homology) indicative of replication slippage.Our data establish that collapsed forks, but not stalled forks, recovered by homologous recombination are prone to replication slippage.We propose that deletions/insertions, mediated by micro-homology, leading to copy number variations during replication stress may arise by progression of error-prone replication forks restarted by homologous recombination.

View Article: PubMed Central - PubMed

Affiliation: Institut Curie, Centre de Recherche, Orsay, France.

ABSTRACT
Homologous recombination is a universal mechanism that allows repair of DNA and provides support for DNA replication. Homologous recombination is therefore a major pathway that suppresses non-homology-mediated genome instability. Here, we report that recovery of impeded replication forks by homologous recombination is error-prone. Using a fork-arrest-based assay in fission yeast, we demonstrate that a single collapsed fork can cause mutations and large-scale genomic changes, including deletions and translocations. Fork-arrest-induced gross chromosomal rearrangements are mediated by inappropriate ectopic recombination events at the site of collapsed forks. Inverted repeats near the site of fork collapse stimulate large-scale genomic changes up to 1,500 times over spontaneous events. We also show that the high accuracy of DNA replication during S-phase is impaired by impediments to fork progression, since fork-arrest-induced mutation is due to erroneous DNA synthesis during recovery of replication forks. The mutations caused are small insertions/duplications between short tandem repeats (micro-homology) indicative of replication slippage. Our data establish that collapsed forks, but not stalled forks, recovered by homologous recombination are prone to replication slippage. The inaccuracy of DNA synthesis does not rely on PCNA ubiquitination or trans-lesion-synthesis DNA polymerases, and it is not counteracted by mismatch repair. We propose that deletions/insertions, mediated by micro-homology, leading to copy number variations during replication stress may arise by progression of error-prone replication forks restarted by homologous recombination.

Show MeSH
Related in: MedlinePlus