53BP1 Protects against CtIP-Dependent Capture of Ectopic Chromosomal Sequences at the Junction of Distant Double-Strand Breaks

DNA double-strand breaks (DSB) are very harmful lesions that can generate genome rearrangements. In this study, we used intrachromosomal reporters to compare both the efficiency and accuracy of end-joining occurring with close (34 bp apart) vs. distant DSBs (3200 bp apart) in human fibroblasts. We showed that a few kb between two intrachromosomal I-SceI-induced DSBs are sufficient to foster deletions and capture/insertions at the junction scar. Captured sequences are mostly coupled to deletions and can be partial duplications of the reporter (i.e., sequences adjacent to the DSB) or insertions of ectopic chromosomal sequences (ECS). Interestingly, silencing 53BP1 stimulates capture/insertions with distant but not with close double-strand ends (DSEs), although deletions were stimulated in both case. This shows that 53BP1 protects both close and distant DSEs from degradation and that the association of unprotection with distance between DSEs favors ECS capture. Reciprocally, silencing CtIP lessens ECS capture both in control and 53BP1-depleted cells. We propose that close ends are immediately/rapidly tethered and ligated, whereas distant ends first require synapsis of the distant DSEs prior to ligation. This "spatio-temporal" gap gives time and space for CtIP to initiate DNA resection, suggesting an involvement of single-stranded DNA tails for ECS capture. We therefore speculate that the resulting single-stranded DNA copies ECS through microhomology-mediated template switching

The Cohesin Complex Prevents the End Joining of Distant DNA Double-Strand Ends

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A threshold of endogenous stress is required to engage cellular response to protect against mutagenesis

International audienceEndogenous stress represents a major source of genome instability, but is in essence difficult to apprehend. Incorporation of labeled radionuclides into DNA constitutes a tractable model to analyze cellular responses to endogenous attacks. Here we show that incorporation of [$^3$H]thymidine into CHO cells generates oxidative-induced mutagenesis, but, with a peak at low doses. Proteomic analysis showed that the cellular response differs between low and high levels of endogenous stress. In particular, these results confirmed the involvement of proteins implicated in redox homeostasis and DNA damage signaling pathways. Induced-mutagenesis was abolished by the anti-oxidant N-acetyl cysteine and plateaued, at high doses, upon exposure to L-buthionine sulfoximine, which represses cellular detoxification. The [$^3$H]thymidine-induced mutation spectrum revealed mostly base substitutions, exhibiting a signature specific for low doses (GC > CG and AT > CG). Consistently, the enzymatic activity of the base excision repair protein APE-1 is induced at only medium or high doses. Collectively, the data reveal that a threshold of endogenous stress must be reached to trigger cellular detoxification and DNA repair programs; below this threshold, the consequences of endogenous stress escape cellular surveillance, leading to high levels of mutagenesis. Therefore, low doses of endogenous local stress can jeopardize genome integrity more efficiently than higher doses

The distance between DSB affects the efficiency of end joining.

<p><b>A.</b> Frequency of end joining in cell lines bearing one or two substrates: 12 independent clones contained one or two of the substrates. Values represent the average ± SEM of at least 5 independent experiments.</p

Accuracy of End Joining on distal or close DSEs.

<p>Accuracy of End Joining on distal or close DSEs.</p

Accuracy of End Joining on distant or close DSEs, upon 53BP1 and/or CtIP depletion.

<p>Accuracy of End Joining on distant or close DSEs, upon 53BP1 and/or CtIP depletion.</p

Slow Replication Fork Velocity of Homologous Recombination-Defective Cells Results from Endogenous Oxidative Stress

International audienceReplications forks are routinely hindered by different endogenous stresses. Because homologous recombination plays a pivotal role in the reactivation of arrested replication forks, defects in homologous recombination reveal the initial endogenous stress(es). Homologous recombination-defective cells consistently exhibit a spontaneously reduced replication speed, leading to mitotic extra centrosomes. Here, we identify oxidative stress as a major endogenous source of replication speed deceleration in homologous recombination-defective cells. The treatment of homologous recombination-defective cells with the antioxidant N-acetyl-cysteine or the maintenance of the cells at low O2 levels (3%) rescues both the replication fork speed, as monitored by single-molecule analysis (molecular combing), and the associated mitotic extra centrosome frequency. Reciprocally, the exposure of wild-type cells to H2O2 reduces the replication fork speed and generates mitotic extra centrosomes. Supplying deoxynucleotide precursors to H2O2-exposed cells rescued the replication speed. Remarkably, treatment with N-acetyl-cysteine strongly expanded the nucleotide pool, accounting for the replication speed rescue. Remarkably, homologous recombination-defective cells exhibit a high level of endogenous reactive oxygen species. Consistently, homologous recombination-defective cells accumulate spontaneous γH2AX or XRCC1 foci that are abolished by treatment with N-acetyl-cysteine or maintenance at 3% O2. Finally, oxidative stress stimulated homologous recombination, which is suppressed by supplying deoxynucleotide precursors. Therefore, the cellular redox status strongly impacts genome duplication and transmission. Oxidative stress should generate replication stress through different mechanisms, including DNA damage and nucleotide pool imbalance. These data highlight the intricacy of endogenous replication and oxidative stresses, which are both evoked during tumorigenesis and senescence initiation, and emphasize the importance of homologous recombination as a barrier against spontaneous genetic instability triggered by the endogenous oxidative/replication stress axis

Origin of large insertions (>45 bp) monitored in the repair of distant DSEs (CD4-3200bp) upon 53BP1 and/or CtIP depletion, in the GC92 cell line.

<p>Origin of large insertions (>45 bp) monitored in the repair of distant DSEs (CD4-3200bp) upon 53BP1 and/or CtIP depletion, in the GC92 cell line.</p

Origin of large insertions (>45 bp) monitored in the repair of distant DSEs (CD4-3200bp) upon 53BP1 depletion in a second cell line (GC49).

<p>Origin of large insertions (>45 bp) monitored in the repair of distant DSEs (CD4-3200bp) upon 53BP1 depletion in a second cell line (GC49).</p

Long insertions are favored at the repair junction of unprotected distant DSEs.

<p><b>A</b>. Impact of 53BP1 and/or CtIP depletion on the size of insertions at the repair sites of distant ends (GC92 cells) or close ends (GCK20 cells) in cells transfected with control siRNA and/ or 53BP1 and/or CtIP siRNAs. For each sample, each dot represents one insertion and the red line represents the median (*: p<0.03, Mann-Whitney test). The green line indicates the threshold of 45 bp that was chosen for sequence BLAST. <b>B.</b> Impact of 53BP1 and CtIP on the frequency of long insertions (≥45 bp). Histograms represent insertions coupled to a deletion event and insertions not coupled to a deletion event. Values represent the mean +/- SEM of at least 3 independent experiments and sequencing of 78 to 190 junction sequences. (*: p = 0.02; **: p<0.005, Mann-Whitney test).</p