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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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Fork recovery by homologous recombination results in replication slippage.A. Serial tenfold-dilutions from indicated strains (t-ura4-dup20-ori associated or not with the RTS1-RFB) were spotted onto the indicated media after cell growth with (Rtf1 -, repressed) or without (Rtf1 +, expressed) thiamine. RTS1-RFB activity is given for each construct and condition. B. Frequency of Ura+ revertants in indicated strains after cell growth with (Rtf1 repressed) or without (Rtf1 expressed) thiamine. The RTS1-RFB activity is given for each construct and condition. Values correspond to the mean of at least three independent experiments and error bars correspond to the standard error of the mean (SEM). C. Rate of replication slippage in the indicated strains and conditions. The rate of Ura+ revertants was calculated using the method of the median from at least 11 independent cultures. Values in brackets indicate the 95% confidence interval. Statistical significance was detected using the nonparametric Mann-Whitney U test. D. Serial tenfold-dilutions from the strains indicated spotted onto the media indicated after cell growth without thiamine. RTS1-RFB activity “–” refers to the strain t-ura4-dup20-ori and “+” refers to the strain t-ura4-dup20<ori. E. Kinetics of Ura+ revertants frequency for the strains indicated as a function of the number of generations after thiamine removal. RTS1-RFB activity “–” refers to the strain t-ura4-dup20-ori and “+” refers to the strain t-ura4-dup20<ori. The values reported are the means of two experiments. F. The rate of replication slippage/generation for the strains indicated with (t-ura4-dup20<ori) or without (t-ura4-dup20-ori) the active RTS1-RFB. The rate was calculated from the slope of the curves presented in panel F. The values reported are means of three independent experiments and error bars correspond to SE.
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pgen-1002976-g004: Fork recovery by homologous recombination results in replication slippage.A. Serial tenfold-dilutions from indicated strains (t-ura4-dup20-ori associated or not with the RTS1-RFB) were spotted onto the indicated media after cell growth with (Rtf1 -, repressed) or without (Rtf1 +, expressed) thiamine. RTS1-RFB activity is given for each construct and condition. B. Frequency of Ura+ revertants in indicated strains after cell growth with (Rtf1 repressed) or without (Rtf1 expressed) thiamine. The RTS1-RFB activity is given for each construct and condition. Values correspond to the mean of at least three independent experiments and error bars correspond to the standard error of the mean (SEM). C. Rate of replication slippage in the indicated strains and conditions. The rate of Ura+ revertants was calculated using the method of the median from at least 11 independent cultures. Values in brackets indicate the 95% confidence interval. Statistical significance was detected using the nonparametric Mann-Whitney U test. D. Serial tenfold-dilutions from the strains indicated spotted onto the media indicated after cell growth without thiamine. RTS1-RFB activity “–” refers to the strain t-ura4-dup20-ori and “+” refers to the strain t-ura4-dup20<ori. E. Kinetics of Ura+ revertants frequency for the strains indicated as a function of the number of generations after thiamine removal. RTS1-RFB activity “–” refers to the strain t-ura4-dup20-ori and “+” refers to the strain t-ura4-dup20<ori. The values reported are the means of two experiments. F. The rate of replication slippage/generation for the strains indicated with (t-ura4-dup20<ori) or without (t-ura4-dup20-ori) the active RTS1-RFB. The rate was calculated from the slope of the curves presented in panel F. The values reported are means of three independent experiments and error bars correspond to SE.

Mentions: To confirm that replication slippage occurs as forks recover, and not behind the fork in the DNA already replicated, we inserted the ura4-dup20 or the ura4-dup22 allele either behind (in the t<ura4-ori configuration) or in front of the RTS1-RFB (in the t-ura4<ori configuration) (Figure 4). This allows the analysis of the same event of replication slippage behind and ahead of collapsed forks. In the t-ura4<ori configuration, induction of the RTS1-RFB resulted in a 8 and 16 fold increases in the replication slippage frequency for the ura4-dup20 and ura4-dup22 alleles, respectively (Figure 4A and 4B). Similar increases in the rate of replication slippages were observed (Figure 4C). In contrast, in the t<ura4-ori background, the frequency of replication slippage was induced by only 2–3 fold by the RTS1-RFB (Figure 4B–4C). These data confirm that DNA located ahead of collapsed forks is more prone to replication slippage than replicated DNA adjacent to arrested forks, further evidence that replication slippage arises during fork recovery.


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)

Fork recovery by homologous recombination results in replication slippage.A. Serial tenfold-dilutions from indicated strains (t-ura4-dup20-ori associated or not with the RTS1-RFB) were spotted onto the indicated media after cell growth with (Rtf1 -, repressed) or without (Rtf1 +, expressed) thiamine. RTS1-RFB activity is given for each construct and condition. B. Frequency of Ura+ revertants in indicated strains after cell growth with (Rtf1 repressed) or without (Rtf1 expressed) thiamine. The RTS1-RFB activity is given for each construct and condition. Values correspond to the mean of at least three independent experiments and error bars correspond to the standard error of the mean (SEM). C. Rate of replication slippage in the indicated strains and conditions. The rate of Ura+ revertants was calculated using the method of the median from at least 11 independent cultures. Values in brackets indicate the 95% confidence interval. Statistical significance was detected using the nonparametric Mann-Whitney U test. D. Serial tenfold-dilutions from the strains indicated spotted onto the media indicated after cell growth without thiamine. RTS1-RFB activity “–” refers to the strain t-ura4-dup20-ori and “+” refers to the strain t-ura4-dup20<ori. E. Kinetics of Ura+ revertants frequency for the strains indicated as a function of the number of generations after thiamine removal. RTS1-RFB activity “–” refers to the strain t-ura4-dup20-ori and “+” refers to the strain t-ura4-dup20<ori. The values reported are the means of two experiments. F. The rate of replication slippage/generation for the strains indicated with (t-ura4-dup20<ori) or without (t-ura4-dup20-ori) the active RTS1-RFB. The rate was calculated from the slope of the curves presented in panel F. The values reported are means of three independent experiments and error bars correspond to SE.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1002976-g004: Fork recovery by homologous recombination results in replication slippage.A. Serial tenfold-dilutions from indicated strains (t-ura4-dup20-ori associated or not with the RTS1-RFB) were spotted onto the indicated media after cell growth with (Rtf1 -, repressed) or without (Rtf1 +, expressed) thiamine. RTS1-RFB activity is given for each construct and condition. B. Frequency of Ura+ revertants in indicated strains after cell growth with (Rtf1 repressed) or without (Rtf1 expressed) thiamine. The RTS1-RFB activity is given for each construct and condition. Values correspond to the mean of at least three independent experiments and error bars correspond to the standard error of the mean (SEM). C. Rate of replication slippage in the indicated strains and conditions. The rate of Ura+ revertants was calculated using the method of the median from at least 11 independent cultures. Values in brackets indicate the 95% confidence interval. Statistical significance was detected using the nonparametric Mann-Whitney U test. D. Serial tenfold-dilutions from the strains indicated spotted onto the media indicated after cell growth without thiamine. RTS1-RFB activity “–” refers to the strain t-ura4-dup20-ori and “+” refers to the strain t-ura4-dup20<ori. E. Kinetics of Ura+ revertants frequency for the strains indicated as a function of the number of generations after thiamine removal. RTS1-RFB activity “–” refers to the strain t-ura4-dup20-ori and “+” refers to the strain t-ura4-dup20<ori. The values reported are the means of two experiments. F. The rate of replication slippage/generation for the strains indicated with (t-ura4-dup20<ori) or without (t-ura4-dup20-ori) the active RTS1-RFB. The rate was calculated from the slope of the curves presented in panel F. The values reported are means of three independent experiments and error bars correspond to SE.
Mentions: To confirm that replication slippage occurs as forks recover, and not behind the fork in the DNA already replicated, we inserted the ura4-dup20 or the ura4-dup22 allele either behind (in the t<ura4-ori configuration) or in front of the RTS1-RFB (in the t-ura4<ori configuration) (Figure 4). This allows the analysis of the same event of replication slippage behind and ahead of collapsed forks. In the t-ura4<ori configuration, induction of the RTS1-RFB resulted in a 8 and 16 fold increases in the replication slippage frequency for the ura4-dup20 and ura4-dup22 alleles, respectively (Figure 4A and 4B). Similar increases in the rate of replication slippages were observed (Figure 4C). In contrast, in the t<ura4-ori background, the frequency of replication slippage was induced by only 2–3 fold by the RTS1-RFB (Figure 4B–4C). These data confirm that DNA located ahead of collapsed forks is more prone to replication slippage than replicated DNA adjacent to arrested forks, further evidence that replication slippage arises during fork recovery.

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