TRIP13 Participates in Immediate-Early Sensing of DNA Strand Breaks and ATM Signaling Amplification through MRE11

Abstract

Thyroid hormone receptor-interacting protein 13 (TRIP13) participates in various regulatory steps related to the cell cycle, such as the mitotic spindle assembly checkpoint and meiotic recombination, possibly by interacting with members of the HORMA domain protein family. Recently, it was reported that TRIP13 could regulate the choice of the DNA repair pathway, i.e., homologous recombination (HR) or nonhomologous end-joining (NHEJ). However, TRIP13 is recruited to DNA damage sites within a few seconds after damage and may therefore have another function in DNA repair other than regulation of the pathway choice. Furthermore, the depletion of TRIP13 inhibited both HR and NHEJ, suggesting that TRIP13 plays other roles besides regulation of choice between HR and NHEJ. To explore the unidentified functions of TRIP13 in the DNA damage response, we investigated its genome-wide interaction partners in the context of DNA damage using quantitative proteomics with proximity labeling. We identified MRE11 as a novel interacting partner of TRIP13. TRIP13 controlled the recruitment of MDC1 to DNA damage sites by regulating the interaction between MDC1 and the MRN complex. Consistently, TRIP13 was involved in ATM signaling amplification. Our study provides new insight into the function of TRIP13 in immediate-early DNA damage sensing and ATM signaling activation.

Keywords: DNA damage response; HORMA domain; MRN complex; TRIP13.

Figure 1

TRIP13 is required for DSB repair. (A) I-SceI-based GFP-reporter assays were performed to measure the contributions of mammalian chromosomal double-strand break repair pathways upon TRIP13 knockdown. TRIP13 knockdown selectively inhibited the HDR and SSA pathways but not NHEJ. DR-GFP, SA-GFP, EJ5-GFP, and EJ2-GFP were used to measure HDR, SSA, classical NHEJ, and alt-NHEJ, respectively [22]. (B) GFP-labeled TRIP13 was rapidly recruited to DNA damage sites induced by UV laser microirradiation. (C) A colony formation assay confirmed that IAA-induced degradation of TRIP13 affected cellular responses to DNA damage induced by IR or HU treatment. The two-tailed unpaired T-test was performed to determine significance (n.s: not significant; ** p  <  0.01; **** p  <  0.0001).

Scheme 1

Downregulated TRIP13 expression and total MDC1 expression were confirmed and DNA damage sensitization under TRIP13 depletion neutral comet. (A) Knockdown of TRIP13 in GFP−reporter U2OS cell lines. (B) IAA-induced TRIP13 degradation for 8 h was confirmed. (C) Total MDC1 expression was not affected under TRIP13 degradation, Mirin treatment and irradiation. (D) Neutral comet assay under TRIP13 degradation with various DNA damage conditions showed the sensitization (* p < 0.05; *** p  <  0.001; **** p  <  0.0001).

Figure 2

MRE11 is a novel interaction partner of TRIP13. (A) A proteomics experiment with BioID2-tagged TRIP13 identified 279 candidate interaction partners. (B) Well-known substrates of TRIP13 (p31comet, MAD2, and CDC20) were labeled by BioID2-TRIP13. (C) Endogenous MRE11 was detected by Strep-AP using Strep-tagged TRIP13. (D) Purified 3xFLAG tagged MRE11 was also detected by in vitro Strep-AP using purified Strep-tagged TRIP13. (E) Biotin pulldown assays of the MRN complex and cofactors (CtIP and ATM) using doxycycline induced BioID2-tagged TRIP13 expressing U-2-OS cells upon CPT treatment for one hour before analysis. (F) FLAG immunoprecipitation through overexpressing both 3xFLAG-TRIP13 and Myc-MRE11 domain deletions series (G) The interactions of MRN complex components with MRE11 were confirmed by immunoprecipitation of MRE11. Depletion of TRIP13 or mirin treatment did not affect these interactions. (H) In vitro MRN endonuclease assays showed that endonuclease activity of the MRN complex was unaffected in the presence of TRIP13 (WT: wild-type, KA: ATP-binding domain mutant, and EQ: ATP hydrolysis mutant). The red arrow indicates the predominant product in the presence of DNA-PKcs and Ku.

Figure 3

Depletion of TRIP13 inhibits MDC1 recruitment to DNA damage sites by decreasing the physical interaction between the MRN complex and MDC1. (A) The chromatin-bound fraction of MDC1 was decreased when TRIP13 was degraded by IAA treatment. We analyzed the cells after one-hour IR irradiation. We also confirmed that TRIP13 depletion did not alter the expression level of MDC1 with whole cell lysates (Scheme 1C). (B) The interaction between MDC1 and MRE11 was reduced upon exposure to IR when TRIP13 was depleted. (C) Formation of MDC1 foci was investigated upon TRIP13 degradation and mirin treatment. We counted the following number of cells for this analysis: 540 cells for control (CON), 354 cells for IR, 594 cells for IAA IR, 530 cells for MIRIN IR, and 488 cells for IAA MIRIN IR. Two-way ANOVA was performed to determine significance (** p  <  0.01; *** p  <  0.001). (D) Formation of phosphor-ATM (S1981) foci was investigated upon TRIP13 degradation and mirin treatment. Two-way ANOVA was performed to determine significance (n.s, not significant; **** p  <  0.0001). (E) TRIP13 knockdown delayed MDC1 recruitment to DNA damage sites. The red lines indicate irradiated region.

Figure 4

TRIP13 enhances HR by regulating ATM downstream signaling and DNA end resection independent of MRE11 exonuclease activity. (A) IR-induced (one-hour irradiation) ATM downstream signaling pathways were downregulated upon TRIP13 degradation with mirin treatment. (B) A quantitative DNA resection assay using the ER−AsiSI system showed that TRIP13 knockdown and mirin treatment inhibited DNA end resection. (C) SCE formation was reduced upon mirin treatment with TRIP13 depletion. (D,E) GFP-reporter assays showed the additive effects of TRIP13 knockdown and mirin treatment on HDR and SSA. The two-tailed unpaired T test was used to determine significance (* p < 0.05; ** p  <  0.01; *** p  <  0.001; n.s, not significant).