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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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Collapsed forks, but not stalled forks, induce replication slippage.A. Left panel: the frequency of Ura+ revertants as a function of time-contact with indicated drugs for the indicated ura4 alleles (single base-substitution, frame-shift, duplication of 20 nt). Right panel: the frequency of Ura+ revertants in response to UV-C irradiation as a function of dose for the indicated ura4 alleles. The values reported are means of two independent experiments. Numbers indicate fold difference in the frequency of Ura+ revertants between the treated and untreated control samples. For ura4 alleles containing base-substitutions or frame-shifts, the mutation event required to obtain Ura+ revertants is indicated on the figure. B. Serial tenfold-dilutions from ura4-dup20 strain spotted onto the media indicated after treatment with MMC or CPT as indicated. C. Frequency of Ura+ revertants after the indicated treatments in the ura4-dup20 strain. DMSO (the vehicle) was used as control for CPT treatment. The values reported are means of at least three independent experiments. Error bars correspond to SEM.
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pgen-1002976-g005: Collapsed forks, but not stalled forks, induce replication slippage.A. Left panel: the frequency of Ura+ revertants as a function of time-contact with indicated drugs for the indicated ura4 alleles (single base-substitution, frame-shift, duplication of 20 nt). Right panel: the frequency of Ura+ revertants in response to UV-C irradiation as a function of dose for the indicated ura4 alleles. The values reported are means of two independent experiments. Numbers indicate fold difference in the frequency of Ura+ revertants between the treated and untreated control samples. For ura4 alleles containing base-substitutions or frame-shifts, the mutation event required to obtain Ura+ revertants is indicated on the figure. B. Serial tenfold-dilutions from ura4-dup20 strain spotted onto the media indicated after treatment with MMC or CPT as indicated. C. Frequency of Ura+ revertants after the indicated treatments in the ura4-dup20 strain. DMSO (the vehicle) was used as control for CPT treatment. The values reported are means of at least three independent experiments. Error bars correspond to SEM.

Mentions: We investigated the effects of replication stress, other than the replication block imposed by the RTS1-RFB, on replication slippage. Strains harbouring ura4− alleles (base-substitutions, −1 frame-shift, and ura4-dup20) were exposed to replication-blocking agents or UV-C-induced DNA damages and the frequency of Ura+ revertants was scored. Three hours of treatment with either the topoisomerase I inhibitor camptothecin (CPT) or mitomycin C (MMC), an inter-strand cross-linking agent (ICls), increased the frequency of Ura+ revertants by 3 to 4 fold in the ura4-dup20 strain (Figure 5A and 5B). At equivalent survival (70–90%), DNA-damages induced by a dose of 100 J/m2 of UV-C did not increase the frequency of Ura+ revertants in the ura4-dup20 strain. Increasing the UV-C dose (150 J/m2) resulted in an increased reversion effect. The other ura4 alleles exhibited an opposite behaviour pattern. As expected, UV-C-induced DNA damages, but not CPT or MMC treatment, increased the frequency of Ura+ revertants of the base-substitution and the −1 frame-shift mutants (Figure 5A). Thus, replication slippage, unlike other point mutations, appears to be a mutation event specifically induced by replication stress.


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)

Collapsed forks, but not stalled forks, induce replication slippage.A. Left panel: the frequency of Ura+ revertants as a function of time-contact with indicated drugs for the indicated ura4 alleles (single base-substitution, frame-shift, duplication of 20 nt). Right panel: the frequency of Ura+ revertants in response to UV-C irradiation as a function of dose for the indicated ura4 alleles. The values reported are means of two independent experiments. Numbers indicate fold difference in the frequency of Ura+ revertants between the treated and untreated control samples. For ura4 alleles containing base-substitutions or frame-shifts, the mutation event required to obtain Ura+ revertants is indicated on the figure. B. Serial tenfold-dilutions from ura4-dup20 strain spotted onto the media indicated after treatment with MMC or CPT as indicated. C. Frequency of Ura+ revertants after the indicated treatments in the ura4-dup20 strain. DMSO (the vehicle) was used as control for CPT treatment. The values reported are means of at least three independent experiments. Error bars correspond to SEM.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3475662&req=5

pgen-1002976-g005: Collapsed forks, but not stalled forks, induce replication slippage.A. Left panel: the frequency of Ura+ revertants as a function of time-contact with indicated drugs for the indicated ura4 alleles (single base-substitution, frame-shift, duplication of 20 nt). Right panel: the frequency of Ura+ revertants in response to UV-C irradiation as a function of dose for the indicated ura4 alleles. The values reported are means of two independent experiments. Numbers indicate fold difference in the frequency of Ura+ revertants between the treated and untreated control samples. For ura4 alleles containing base-substitutions or frame-shifts, the mutation event required to obtain Ura+ revertants is indicated on the figure. B. Serial tenfold-dilutions from ura4-dup20 strain spotted onto the media indicated after treatment with MMC or CPT as indicated. C. Frequency of Ura+ revertants after the indicated treatments in the ura4-dup20 strain. DMSO (the vehicle) was used as control for CPT treatment. The values reported are means of at least three independent experiments. Error bars correspond to SEM.
Mentions: We investigated the effects of replication stress, other than the replication block imposed by the RTS1-RFB, on replication slippage. Strains harbouring ura4− alleles (base-substitutions, −1 frame-shift, and ura4-dup20) were exposed to replication-blocking agents or UV-C-induced DNA damages and the frequency of Ura+ revertants was scored. Three hours of treatment with either the topoisomerase I inhibitor camptothecin (CPT) or mitomycin C (MMC), an inter-strand cross-linking agent (ICls), increased the frequency of Ura+ revertants by 3 to 4 fold in the ura4-dup20 strain (Figure 5A and 5B). At equivalent survival (70–90%), DNA-damages induced by a dose of 100 J/m2 of UV-C did not increase the frequency of Ura+ revertants in the ura4-dup20 strain. Increasing the UV-C dose (150 J/m2) resulted in an increased reversion effect. The other ura4 alleles exhibited an opposite behaviour pattern. As expected, UV-C-induced DNA damages, but not CPT or MMC treatment, increased the frequency of Ura+ revertants of the base-substitution and the −1 frame-shift mutants (Figure 5A). Thus, replication slippage, unlike other point mutations, appears to be a mutation event specifically induced by replication stress.

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