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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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Related in: MedlinePlus

Conditional replication fork-arrest assays.A. Diagrams of fork-arrest constructs. Centromere-proximal and telomere-proximal regions are represented in black and grey, respectively. Strong or putative replication origins (ori) and the centromere are indicated by yellow, green and black circles, respectively. Blues arrows indicate the polarity of the RTS1-RFB. The ura4+ gene is indicated in red and the arrow indicates its direction of transcription. Representations of the primary arrested fork structure are given for each construct. The name of each fork-arrest construct is given using the following nomenclature: “t” and “ori” refer to the telomere and the replication origin 3006/7, respectively; “<“ and ”>” indicate the RTS1-barrier and its polarity (< blocks replication forks moving from the ori3006/7 towards the telomere, and > blocks replication forks moving from the telomere towards the origin 3006/7. B. Diagrams of replication intermediates (RIs) within the AseI fragment analysed by 2DGE (top panel). Representative RIs analysed by 2DGE in indicated strains in OFF (Rtf1 being repressed) and ON (Rtf1 being expressed) conditions. Signal corresponding to arrested forks, joints-molecules (JMs) and termination structures are indicated by black, red and green arrows, respectively. Note that the t>ura4-ori construct does not result in a strong fork arrest as the RTS1-RFB is not orientated in the main direction of replication (see text for details).
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pgen-1002976-g001: Conditional replication fork-arrest assays.A. Diagrams of fork-arrest constructs. Centromere-proximal and telomere-proximal regions are represented in black and grey, respectively. Strong or putative replication origins (ori) and the centromere are indicated by yellow, green and black circles, respectively. Blues arrows indicate the polarity of the RTS1-RFB. The ura4+ gene is indicated in red and the arrow indicates its direction of transcription. Representations of the primary arrested fork structure are given for each construct. The name of each fork-arrest construct is given using the following nomenclature: “t” and “ori” refer to the telomere and the replication origin 3006/7, respectively; “<“ and ”>” indicate the RTS1-barrier and its polarity (< blocks replication forks moving from the ori3006/7 towards the telomere, and > blocks replication forks moving from the telomere towards the origin 3006/7. B. Diagrams of replication intermediates (RIs) within the AseI fragment analysed by 2DGE (top panel). Representative RIs analysed by 2DGE in indicated strains in OFF (Rtf1 being repressed) and ON (Rtf1 being expressed) conditions. Signal corresponding to arrested forks, joints-molecules (JMs) and termination structures are indicated by black, red and green arrows, respectively. Note that the t>ura4-ori construct does not result in a strong fork arrest as the RTS1-RFB is not orientated in the main direction of replication (see text for details).

Mentions: We generated fork arrest constructs by manipulating the polar RTS1-RFB (Figure 1A). We introduced the RTS1 sequence on the centromere-proximal (cen-proximal) side of the ura4 locus, 5 kb away from the strong replication origin (ori) 3006/7 on chromosome III. This created the t-ura4<ori locus, in which “t” and “ori” refer to the telomere and the origin 3006/7, respectively; and “<” and“ >”refer to the RTS1-barrier and its polarity that is whether it blocks replication forks travelling from the ori 3006/7 towards the telomere or forks travelling from the telomere towards the ori 3006/7, respectively. We previously confirmed that forks moving from ori 3006/7 towards the telomere (tel) are efficiently blocked by the RTS1-RFB at the t-ura4<ori locus [35]. In this model system, fork arrest is activated by inducing the expression of rtf1+ gene that is under control of the thiamine repressible promoter nmt41. Thus, the RTS1-RFB is inactivated by adding thiamine to the media and it is activated in thiamine-free media. Efficient induction of Rtf1 expression requires incubation for 12–16 hours in thiamine-free media. Replication intermediates were analysed by native 2-dimensional gel electrophoresis (2DGE). In conditions of Rtf1 expression, more than 95% of replication forks were blocked by the RTS1-RFB at the t-ura4<ori locus (see black arrow on Figure 1B, t-ura4<ori ON). Arrested forks were not detected without Rtf1 induction (Figure 1B, t-ura4<ori OFF) [20].


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)

Conditional replication fork-arrest assays.A. Diagrams of fork-arrest constructs. Centromere-proximal and telomere-proximal regions are represented in black and grey, respectively. Strong or putative replication origins (ori) and the centromere are indicated by yellow, green and black circles, respectively. Blues arrows indicate the polarity of the RTS1-RFB. The ura4+ gene is indicated in red and the arrow indicates its direction of transcription. Representations of the primary arrested fork structure are given for each construct. The name of each fork-arrest construct is given using the following nomenclature: “t” and “ori” refer to the telomere and the replication origin 3006/7, respectively; “<“ and ”>” indicate the RTS1-barrier and its polarity (< blocks replication forks moving from the ori3006/7 towards the telomere, and > blocks replication forks moving from the telomere towards the origin 3006/7. B. Diagrams of replication intermediates (RIs) within the AseI fragment analysed by 2DGE (top panel). Representative RIs analysed by 2DGE in indicated strains in OFF (Rtf1 being repressed) and ON (Rtf1 being expressed) conditions. Signal corresponding to arrested forks, joints-molecules (JMs) and termination structures are indicated by black, red and green arrows, respectively. Note that the t>ura4-ori construct does not result in a strong fork arrest as the RTS1-RFB is not orientated in the main direction of replication (see text for details).
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1002976-g001: Conditional replication fork-arrest assays.A. Diagrams of fork-arrest constructs. Centromere-proximal and telomere-proximal regions are represented in black and grey, respectively. Strong or putative replication origins (ori) and the centromere are indicated by yellow, green and black circles, respectively. Blues arrows indicate the polarity of the RTS1-RFB. The ura4+ gene is indicated in red and the arrow indicates its direction of transcription. Representations of the primary arrested fork structure are given for each construct. The name of each fork-arrest construct is given using the following nomenclature: “t” and “ori” refer to the telomere and the replication origin 3006/7, respectively; “<“ and ”>” indicate the RTS1-barrier and its polarity (< blocks replication forks moving from the ori3006/7 towards the telomere, and > blocks replication forks moving from the telomere towards the origin 3006/7. B. Diagrams of replication intermediates (RIs) within the AseI fragment analysed by 2DGE (top panel). Representative RIs analysed by 2DGE in indicated strains in OFF (Rtf1 being repressed) and ON (Rtf1 being expressed) conditions. Signal corresponding to arrested forks, joints-molecules (JMs) and termination structures are indicated by black, red and green arrows, respectively. Note that the t>ura4-ori construct does not result in a strong fork arrest as the RTS1-RFB is not orientated in the main direction of replication (see text for details).
Mentions: We generated fork arrest constructs by manipulating the polar RTS1-RFB (Figure 1A). We introduced the RTS1 sequence on the centromere-proximal (cen-proximal) side of the ura4 locus, 5 kb away from the strong replication origin (ori) 3006/7 on chromosome III. This created the t-ura4<ori locus, in which “t” and “ori” refer to the telomere and the origin 3006/7, respectively; and “<” and“ >”refer to the RTS1-barrier and its polarity that is whether it blocks replication forks travelling from the ori 3006/7 towards the telomere or forks travelling from the telomere towards the ori 3006/7, respectively. We previously confirmed that forks moving from ori 3006/7 towards the telomere (tel) are efficiently blocked by the RTS1-RFB at the t-ura4<ori locus [35]. In this model system, fork arrest is activated by inducing the expression of rtf1+ gene that is under control of the thiamine repressible promoter nmt41. Thus, the RTS1-RFB is inactivated by adding thiamine to the media and it is activated in thiamine-free media. Efficient induction of Rtf1 expression requires incubation for 12–16 hours in thiamine-free media. Replication intermediates were analysed by native 2-dimensional gel electrophoresis (2DGE). In conditions of Rtf1 expression, more than 95% of replication forks were blocked by the RTS1-RFB at the t-ura4<ori locus (see black arrow on Figure 1B, t-ura4<ori ON). Arrested forks were not detected without Rtf1 induction (Figure 1B, t-ura4<ori OFF) [20].

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