SDE2 integrates into the TIMELESS-TIPIN complex to protect stalled replication forks

Protecting replication fork integrity during DNA replication is essential for maintaining genome stability. Here, we report that SDE2, a PCNA-associated protein, plays a key role in maintaining active replication and counteracting replication stress by regulating the replication fork protection complex (FPC). SDE2 directly interacts with the FPC component TIMELESS (TIM) and enhances its stability, thereby aiding TIM localization to replication forks and the coordination of replisome progression. Like TIM deficiency, knockdown of SDE2 leads to impaired fork progression and stalled fork recovery, along with a failure to activate CHK1 phosphorylation. Moreover, loss of SDE2 or TIM results in an excessive MRE11-dependent degradation of reversed forks. Together, our study uncovers an essential role for SDE2 in maintaining genomic integrity by stabilizing the FPC and describes a new role for TIM in protecting stalled replication forks. We propose that TIM-mediated fork protection may represent a way to cooperate with BRCA-dependent fork stabilization.


Statistics
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Replication
All cellular and biochemical experiments were repeated as independent biological replicates. Western blotting, DNA combing, immunofluorescence, DNA comet assay, survival assay, and cell cycle analysis were performed at least twice independently, mostly three times. Survival assays were technically duplicated in each set. We confirmed that the repeated experiments showed similar results and all attempts to reproduce results were successful.
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For Western blotting, DNA combing, immunofluorescence, DNA comet assay, survival assay, and cell cycle analysis, data of different groups were collected while no blinding was used since blinding was not relevant for this kind of experiments in which researchers are aware of particular experimental conditions. When data were analyzed by software, researchers were generally not blinded to group allocation.

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Flow Cytometry
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To label replicating cells, siRNA-transfected cells were incubated with 10 μM EdU (Thermo Fisher) for 30 min before harvest. Harvested cells were fixed with 4% paraformaldehyde for 15 min at RT, permeabilized by saponin-based permeabilization buffer (Thermo Fisher) for 15 min, and subjected to EdU-click reaction using Alexa Fluor 488 picolyl azide and click-iT Plus EdU flow cytometry assay kit (Thermo Fisher) following manufacturer's protocol. Cells were washed once and resuspended

Instrument
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