Limits...
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.

Show MeSH

Related in: MedlinePlus

Fork-arrest induces mutations.A. Effect of intra- and inter-chromosomal recombination between RTS1 repeats on fork-arrest-induced mutation rates (base-substitutions, frame-shifts and small insertions or deletions between short tandem repeats). RTS1-RFB activity and ura4 location with respect to the RFB are given for each construct. The % of mutation events, as determined by the PCR assay and sequencing, was used to balance the rate of ura4 loss. Then, the RFB-induced mutation 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 SE. (Refer to Figure S2 for corresponding rates of mutation when Rtf1 is expressed or not). B. Rate of mutation for indicated strains; ON and OFF refers to the RTS1-RFB being active or not, respectively. The % of mutation events, as determined by the PCR assay and sequencing, was used to balance the rate of ura4 loss. The values reported are means of at least 3 independent median rates. Error bars correspond to SE. Statistically significant fold differences in mutation rates between the “OFF” and “ON” conditions are indicated with an *. C. Spectra of mutation events in indicated strains upon RFB induction (refer to Table 2 for exact numbers and to Figure S3 for mapping of deletions/duplications and their features). D. Strains harbouring the ura4 alleles with a single base-substitution or frame-shift or duplication of 20 or 22 nt, together with the RTS1-RFB in the t-ura4<ori configuration were streaked onto the indicated media after cell growth with (RFB “OFF”) or without (RFB “ON”) thiamine. The bottom diagram indicates strain positions and the mutation events required to obtain Ura+ revertants. E. PCR analysis of Ura+ revertants isolated from the t-ura4-dup20<ori strain (duplication of 20 nt in ura4) after RFB induction. With the primers used, a 106 bp fragment is amplified from the ura4+ strain and a 126 bp fragment is amplified from the t-ura4-dup20<ori strain. F. Sequence alignments of ura4-dup22, ura4-dup20, ura4+ alleles and corresponding Ura+ revertants. Micro-homologies are indicated in grey and duplicated sequences are underlined in black. The phenotype of each allele is indicated on the figure. G. Map of deletion and duplication events within the ura4 ORF.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3475662&req=5

pgen-1002976-g003: Fork-arrest induces mutations.A. Effect of intra- and inter-chromosomal recombination between RTS1 repeats on fork-arrest-induced mutation rates (base-substitutions, frame-shifts and small insertions or deletions between short tandem repeats). RTS1-RFB activity and ura4 location with respect to the RFB are given for each construct. The % of mutation events, as determined by the PCR assay and sequencing, was used to balance the rate of ura4 loss. Then, the RFB-induced mutation 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 SE. (Refer to Figure S2 for corresponding rates of mutation when Rtf1 is expressed or not). B. Rate of mutation for indicated strains; ON and OFF refers to the RTS1-RFB being active or not, respectively. The % of mutation events, as determined by the PCR assay and sequencing, was used to balance the rate of ura4 loss. The values reported are means of at least 3 independent median rates. Error bars correspond to SE. Statistically significant fold differences in mutation rates between the “OFF” and “ON” conditions are indicated with an *. C. Spectra of mutation events in indicated strains upon RFB induction (refer to Table 2 for exact numbers and to Figure S3 for mapping of deletions/duplications and their features). D. Strains harbouring the ura4 alleles with a single base-substitution or frame-shift or duplication of 20 or 22 nt, together with the RTS1-RFB in the t-ura4<ori configuration were streaked onto the indicated media after cell growth with (RFB “OFF”) or without (RFB “ON”) thiamine. The bottom diagram indicates strain positions and the mutation events required to obtain Ura+ revertants. E. PCR analysis of Ura+ revertants isolated from the t-ura4-dup20<ori strain (duplication of 20 nt in ura4) after RFB induction. With the primers used, a 106 bp fragment is amplified from the ura4+ strain and a 126 bp fragment is amplified from the t-ura4-dup20<ori strain. F. Sequence alignments of ura4-dup22, ura4-dup20, ura4+ alleles and corresponding Ura+ revertants. Micro-homologies are indicated in grey and duplicated sequences are underlined in black. The phenotype of each allele is indicated on the figure. G. Map of deletion and duplication events within the ura4 ORF.

Mentions: We analysed the effects of collapsed forks on the mutation rate. We sequenced the ura4 coding sequence from 5-FOAR isolated cells and identified base-substitutions, frame-shifts and small insertions and duplications between short tandem repeats (Table 2). A single collapsed fork in the t-ura4<ori strain increased the overall mutation rate up to 10 times over spontaneous events (Figure 3A, p = 0.003). Similar increases in the overall mutation rate were found for the strains with IRs near the arrested fork and those with RTS1 deleted from chromosome II (Figure 3A and Figure S2A). Thus, fork-arrest-induced mutation is not mediated by inappropriate ectopic recombination. Induction of the RTS1-RFB in the t<ura4-ori strain did not increase the mutation rate of the ura4 gene. Thus, as for GCRs, replicated regions behind arrested forks are not prone to mutation. This observation rules out the hypothesis that fork-arrest-induced mutation is a consequence of the accumulation of damaged single-stranded DNA behind collapsed forks (see discussion). Our data suggest that recovery from collapsed forks results in error-prone DNA-synthesis.


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-arrest induces mutations.A. Effect of intra- and inter-chromosomal recombination between RTS1 repeats on fork-arrest-induced mutation rates (base-substitutions, frame-shifts and small insertions or deletions between short tandem repeats). RTS1-RFB activity and ura4 location with respect to the RFB are given for each construct. The % of mutation events, as determined by the PCR assay and sequencing, was used to balance the rate of ura4 loss. Then, the RFB-induced mutation 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 SE. (Refer to Figure S2 for corresponding rates of mutation when Rtf1 is expressed or not). B. Rate of mutation for indicated strains; ON and OFF refers to the RTS1-RFB being active or not, respectively. The % of mutation events, as determined by the PCR assay and sequencing, was used to balance the rate of ura4 loss. The values reported are means of at least 3 independent median rates. Error bars correspond to SE. Statistically significant fold differences in mutation rates between the “OFF” and “ON” conditions are indicated with an *. C. Spectra of mutation events in indicated strains upon RFB induction (refer to Table 2 for exact numbers and to Figure S3 for mapping of deletions/duplications and their features). D. Strains harbouring the ura4 alleles with a single base-substitution or frame-shift or duplication of 20 or 22 nt, together with the RTS1-RFB in the t-ura4<ori configuration were streaked onto the indicated media after cell growth with (RFB “OFF”) or without (RFB “ON”) thiamine. The bottom diagram indicates strain positions and the mutation events required to obtain Ura+ revertants. E. PCR analysis of Ura+ revertants isolated from the t-ura4-dup20<ori strain (duplication of 20 nt in ura4) after RFB induction. With the primers used, a 106 bp fragment is amplified from the ura4+ strain and a 126 bp fragment is amplified from the t-ura4-dup20<ori strain. F. Sequence alignments of ura4-dup22, ura4-dup20, ura4+ alleles and corresponding Ura+ revertants. Micro-homologies are indicated in grey and duplicated sequences are underlined in black. The phenotype of each allele is indicated on the figure. G. Map of deletion and duplication events within the ura4 ORF.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1002976-g003: Fork-arrest induces mutations.A. Effect of intra- and inter-chromosomal recombination between RTS1 repeats on fork-arrest-induced mutation rates (base-substitutions, frame-shifts and small insertions or deletions between short tandem repeats). RTS1-RFB activity and ura4 location with respect to the RFB are given for each construct. The % of mutation events, as determined by the PCR assay and sequencing, was used to balance the rate of ura4 loss. Then, the RFB-induced mutation 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 SE. (Refer to Figure S2 for corresponding rates of mutation when Rtf1 is expressed or not). B. Rate of mutation for indicated strains; ON and OFF refers to the RTS1-RFB being active or not, respectively. The % of mutation events, as determined by the PCR assay and sequencing, was used to balance the rate of ura4 loss. The values reported are means of at least 3 independent median rates. Error bars correspond to SE. Statistically significant fold differences in mutation rates between the “OFF” and “ON” conditions are indicated with an *. C. Spectra of mutation events in indicated strains upon RFB induction (refer to Table 2 for exact numbers and to Figure S3 for mapping of deletions/duplications and their features). D. Strains harbouring the ura4 alleles with a single base-substitution or frame-shift or duplication of 20 or 22 nt, together with the RTS1-RFB in the t-ura4<ori configuration were streaked onto the indicated media after cell growth with (RFB “OFF”) or without (RFB “ON”) thiamine. The bottom diagram indicates strain positions and the mutation events required to obtain Ura+ revertants. E. PCR analysis of Ura+ revertants isolated from the t-ura4-dup20<ori strain (duplication of 20 nt in ura4) after RFB induction. With the primers used, a 106 bp fragment is amplified from the ura4+ strain and a 126 bp fragment is amplified from the t-ura4-dup20<ori strain. F. Sequence alignments of ura4-dup22, ura4-dup20, ura4+ alleles and corresponding Ura+ revertants. Micro-homologies are indicated in grey and duplicated sequences are underlined in black. The phenotype of each allele is indicated on the figure. G. Map of deletion and duplication events within the ura4 ORF.
Mentions: We analysed the effects of collapsed forks on the mutation rate. We sequenced the ura4 coding sequence from 5-FOAR isolated cells and identified base-substitutions, frame-shifts and small insertions and duplications between short tandem repeats (Table 2). A single collapsed fork in the t-ura4<ori strain increased the overall mutation rate up to 10 times over spontaneous events (Figure 3A, p = 0.003). Similar increases in the overall mutation rate were found for the strains with IRs near the arrested fork and those with RTS1 deleted from chromosome II (Figure 3A and Figure S2A). Thus, fork-arrest-induced mutation is not mediated by inappropriate ectopic recombination. Induction of the RTS1-RFB in the t<ura4-ori strain did not increase the mutation rate of the ura4 gene. Thus, as for GCRs, replicated regions behind arrested forks are not prone to mutation. This observation rules out the hypothesis that fork-arrest-induced mutation is a consequence of the accumulation of damaged single-stranded DNA behind collapsed forks (see discussion). Our data suggest that recovery from collapsed forks results in error-prone DNA-synthesis.

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