BLM Deficiency Is Not Associated with Sensitivity to Hydroxyurea-Induced Replication Stress

Bloom's syndrome (BS) displays one of the strongest known correlations between chromosomal instability and a high risk of cancer at an early age. BS cells combine a reduced average fork velocity with constitutive endogenous replication stress. However, the response of BS cells to replication stress induced by hydroxyurea (HU), which strongly slows the progression of replication forks, remains unclear due to publication of conflicting results. Using two different cellular models of BS, we showed that BLM deficiency is not associated with sensitivity to HU, in terms of clonogenic survival, DSB generation, and SCE induction. We suggest that surviving BLM-deficient cells are selected on the basis of their ability to deal with an endogenous replication stress induced by replication fork slowing, resulting in insensitivity to HU-induced replication stress

Aire-dependent genes undergo Clp1-mediated 3'UTR shortening associated with higher transcript stability in the thymus

International audienceThe ability of the immune system to avoid autoimmune disease relies on tolerization of thymocytes to self-antigens whose expression and presentation by thymic medullary epithelial cells (mTECs) is controlled predominantly by Aire at the transcriptional level and possibly regulated at other unrecognized levels. Aire-sensitive gene expression is influenced by several molecular factors, some of which belong to the 3’end processing complex, suggesting they might impact transcript stability and levels through an effect on 3’UTR shortening. We discovered that Aire-sensitive genes display a pronounced preference for short-3’UTR transcript isoforms in mTECs, a feature preceding Aire’s expression and correlated with the preferential selection of proximal polyA sites by the 3’end processing complex. Through an RNAi screen and generation of a lentigenic mouse, we found that one factor, Clp1, promotes 3’UTR shortening associated with higher transcript stability and expression of Aire-sensitive genes, revealing a post-transcriptional level of control of Aire-activated expression in mTECs

Recovery of Arrested Replication Forks by Homologous Recombination Is Error-Prone

<div><p>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.</p> </div

Conditional replication fork-arrest assays.

<p>A. Diagrams of fork-arrest constructs. <i>Centromere</i>-proximal and <i>telomere</i>-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 <i>RTS1</i>-RFB. The <i>ura4⁺</i> 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 <i>RTS1</i>-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 A<i>se</i>I 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 <i>t>ura4-ori</i> construct does not result in a strong fork arrest as the <i>RTS1</i>-RFB is not orientated in the main direction of replication (see text for details).</p

Model of replication-stress-induced genetic instability at collapsed forks.

<p>Collapsed forks might arise from torsional stress, fork breakage (<i>i.e.</i> at nick, ICLs), or proteins tightly-bound to DNA. Replisome disassembly at collapsed forks may favour the unwinding of the nascent strand on which Rad51 nucleates. At this initial stage of fork resumption by recombination, homology-driven template exchange can promote intra- or inter-recombination resulting in GCRs. Fork recovery by recombination overcomes the initial replication block and allows an inaccurate replisome to form (see text for details).</p

Fork recovery by homologous recombination results in replication slippage.

<p>A. Serial tenfold-dilutions from indicated strains (<i>t-ura4-dup20-ori</i> associated or not with the <i>RTS1</i>-RFB) were spotted onto the indicated media after cell growth with (Rtf1 -, repressed) or without (Rtf1 +, expressed) thiamine. <i>RTS1</i>-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 <i>RTS1</i>-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. <i>RTS1</i>-RFB activity “–” refers to the strain <i>t-ura4-dup20-ori</i> and “+” refers to the strain <i>t-ura4-dup20. E. Kinetics of Ura⁺ revertants frequency for the strains indicated as a function of the number of generations after thiamine removal. <i>RTS1</i>-RFB activity “–” refers to the strain <i>t-ura4-dup20-ori</i> and “+” refers to the strain <i>t-ura4-dup20. The values reported are the means of two experiments. F. The rate of replication slippage/generation for the strains indicated with (<i>t-ura4-dup20) or without (<i>t-ura4-dup20-ori</i>) the active <i>RTS1</i>-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.</i></i></i></p

A single fork-arrest induces GCRs that are stimulated by inverted repeats near the site of fork arrest.

<p>A. Diagrams of chromosome II containing or not the <i>RTS1</i> sequence (blue arrow or <i>RTS1-d</i>) and of chromosome III containing <i>ura4⁺</i> alone or associated with <i>RTS1-</i>RFB constructs. The <i>RTS1</i> sequence maps near the <i>mat1</i> locus where it helps to ensure unidirectional replication <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002976#pgen.1002976-Dalgaard1" target="_blank">[62]</a>. Primers used for amplifying the 1 Kb <i>ura4</i> fragment or the 650 bp <i>rng3</i> 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-FOA^R colonies of the indicated strains; ON and OFF refers to the <i>RTS1</i>-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 <i>RTS1</i> repeats on fork-arrest-induced genomic deletion. <i>RTS1</i>-RFB activity and <i>ura4</i> 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 <i>ura4</i> 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 <i>rqh1-d</i> and the wild-type strains are indicated with an *. E. Representative PCR amplifications from 5-FOA^R colonies of the <i>rqh1-d t-ura4 strain, as described for panel B. (Refer to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002976#pgen.1002976.s001" target="_blank">Figure S1</a> for corresponding rates of deletion and translocation when Rtf1 is expressed or not).</i></p

Rates of <i>ura4</i> loss (including genomic deletion, translocation, and mutation events), calculated using the method of the median.

a<p> the following nomenclature is used: “t” and “ori” refer to the telomere and the replication origin 3006/7, respectively; “<“ and ”>” indicate the <i>RTS1</i>-RFB 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).</p>b<p> event/cell/division ×10⁻⁸ ± standard error. Values are means of at least 3 independent rates.</p>c<p> statistical significance was determined using the nonparametric Mann-Whitney U test.</p

Collapsed forks, but not stalled forks, induce replication slippage.

<p>A. Left panel: the frequency of Ura⁺ revertants as a function of time-contact with indicated drugs for the indicated <i>ura4</i> 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 <i>ura4</i> 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 <i>ura4</i> 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 <i>ura4-dup20</i> 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 <i>ura4-dup20</i> 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.</p