The homologous recombination component EEPD1 is required for genome stability in response to developmental stress of vertebrate embryogenesis

Chun, Changzoon; Wu, Yuehan; Lee, Suk‐Hee; Williamson, Elizabeth A.; Reinert, Brian L.; Jaiswal, Aruna S.; Nickoloff, Jac A.; Hromas, Robert

doi:10.1080/15384101.2016.1151585

Cited by 18 publications

(22 citation statements)

References 40 publications

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“…Indeed, there is a great deal of evidence that cells will bypass DNA lesions to maintain replication rates [ 41 , 47 ]. We and others have found that even delaying fork repair by 15–30 min can have lethal consequences [ 17 , 19 , 35 , 47 ]. The data presented here suggest a model in which an EEPD1-cleaved replication fork repair intermediate is rapidly recognized by the cell as toxic, and in the absence of BRCA1 and RAD52, the free DS ends are shunted toward the aNHEJ repair pathway.…”

Section: Discussionmentioning

confidence: 99%

“…There are two ways to create this DS end: fork reversal to a chicken foot structure, or fork cleavage by a structure-specific nuclease [ 14 – 16 ]. We previously reported that the 5′ endonuclease EEPD1 could cleave stalled replication forks, initiate EXO1-mediated 5′ end resection, and promote repair of HR replication forks independent of BRCA1 [ 17 – 19 ]. However, BRCA1/2-mutant cancer cells lack HR, and how these cells repair stalled replication forks has been an unresolved issue.…”

Section: Introductionmentioning

confidence: 99%

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The endonuclease EEPD1 mediates synthetic lethality in RAD52-depleted BRCA1 mutant breast cancer cells

et al. 2017

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BackgroundProper repair and restart of stressed replication forks requires intact homologous recombination (HR). HR at stressed replication forks can be initiated by the 5′ endonuclease EEPD1, which cleaves the stalled replication fork. Inherited or acquired defects in HR, such as mutations in breast cancer susceptibility protein-1 (BRCA1) or BRCA2, predispose to cancer, including breast and ovarian cancers. In order for these HR-deficient tumor cells to proliferate, they become addicted to a bypass replication fork repair pathway mediated by radiation repair protein 52 (RAD52). Depleting RAD52 can cause synthetic lethality in BRCA1/2 mutant cancers by an unknown molecular mechanism.MethodsWe hypothesized that cleavage of stressed replication forks by EEPD1 generates a fork repair intermediate that is toxic when HR-deficient cells cannot complete repair with the RAD52 bypass pathway. To test this hypothesis, we applied cell survival assays, immunofluorescence staining, DNA fiber and western blot analyses to look at the correlation between cell survival and genome integrity in control, EEPD1, RAD52 and EEPD1/RAD52 co-depletion BRCA1-deficient breast cancer cells.ResultsOur data show that depletion of EEPD1 suppresses synthetic lethality, genome instability, mitotic catastrophe, and hypersensitivity to stress of replication of RAD52-depleted, BRCA1 mutant breast cancer cells. Without HR and the RAD52-dependent backup pathway, the BRCA1 mutant cancer cells depleted of EEPD1 skew to the alternative non-homologous end-joining DNA repair pathway for survival.ConclusionThis study indicates that the mechanism of synthetic lethality in RAD52-depleted BRCA1 mutant cancer cells depends on the endonuclease EEPD1. The data imply that EEPD1 cleavage of stressed replication forks may result in a toxic intermediate when replication fork repair cannot be completed.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-017-0912-8) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The endonuclease EEPD1 mediates synthetic lethality in RAD52-depleted BRCA1 mutant breast cancer cells

et al. 2017

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“…Next to our study, which is the first to identify EEPD1 as an LXR target and ascribe it a function in cellular sterol homeostasis, 2 other studies from the Hromas group recently reported that EEPD1 has a function in DNA repair in the nucleus. 49 , 50 Wu et al 49 compellingly demonstrated that loss of EEPD1 in several cell types facilitates repair of stressed replication forks induced by DNA-damaging chemicals and that it does so by promoting homologous recombination. In our studies, we have not observed localization of EEPD1 in the nucleus under any of the conditions evaluated.…”

Section: Discussionmentioning

confidence: 99%

EEPD1 Is a Novel LXR Target Gene in Macrophages Which Regulates ABCA1 Abundance and Cholesterol Efflux

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Koenis

Scheij

et al. 2017

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“…Increased IdU:CldU ratios indicate reduced CldU incorporation after release from stress, i.e. delayed fork restart and/or slower progression ( 69 ). IdU is incorporated prior to HU treatment, so we pooled IdU fiber length measurements from treated and untreated cultures and found that replication speed is not reduced in Metnase or EEPD1 KO cells ( Supplementary Figure S7 ).…”

Section: Resultsmentioning

confidence: 99%

Distinct roles of structure-specific endonucleases EEPD1 and Metnase in replication stress responses

et al. 2020

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Accurate DNA replication and segregation are critical for maintaining genome integrity and suppressing cancer. Metnase and EEPD1 are DNA damage response (DDR) proteins frequently dysregulated in cancer and implicated in cancer etiology and tumor response to genotoxic chemo- and radiotherapy. Here, we examine the DDR in human cell lines with CRISPR/Cas9 knockout of Metnase or EEPD1. The knockout cell lines exhibit slightly slower growth rates, significant hypersensitivity to replication stress, increased genome instability and distinct alterations in DDR signaling. Metnase and EEPD1 are structure-specific nucleases. EEPD1 is recruited to and cleaves stalled forks to initiate fork restart by homologous recombination. Here, we demonstrate that Metnase is also recruited to stalled forks where it appears to dimethylate histone H3 lysine 36 (H3K36me2), raising the possibility that H3K36me2 promotes DDR factor recruitment or limits nucleosome eviction to protect forks from nucleolytic attack. We show that stalled forks are cleaved normally in the absence of Metnase, an important and novel result because a prior study indicated that Metnase nuclease is important for timely fork restart. A double knockout was as sensitive to etoposide as either single knockout, suggesting a degree of epistasis between Metnase and EEPD1. We propose that EEPD1 initiates fork restart by cleaving stalled forks, and that Metnase may promote fork restart by processing homologous recombination intermediates and/or inducing H3K36me2 to recruit DDR factors. By accelerating fork restart, Metnase and EEPD1 reduce the chance that stalled replication forks will adopt toxic or genome-destabilizing structures, preventing genome instability and cancer. Metnase and EEPD1 are overexpressed in some cancers and thus may also promote resistance to genotoxic therapeutics.

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The homologous recombination component EEPD1 is required for genome stability in response to developmental stress of vertebrate embryogenesis

Cited by 18 publications

References 40 publications

The endonuclease EEPD1 mediates synthetic lethality in RAD52-depleted BRCA1 mutant breast cancer cells

The endonuclease EEPD1 mediates synthetic lethality in RAD52-depleted BRCA1 mutant breast cancer cells

EEPD1 Is a Novel LXR Target Gene in Macrophages Which Regulates ABCA1 Abundance and Cholesterol Efflux

Distinct roles of structure-specific endonucleases EEPD1 and Metnase in replication stress responses

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