FKBP25 participates in DNA double-strand break repair

Abstract

FK506-binding proteins (FKBPs) alter the conformation of proteins via cis-trans isomerization of prolyl-peptide bonds. While this activity can be demonstrated in vitro, the intractability of detecting prolyl isomerization events in cells has limited our understanding of the biological processes regulated by FKBPs. Here we report that FKBP25 is an active participant in the repair of DNA double-strand breaks (DSBs). FKBP25 influences DSB repair pathway choice by promoting homologous recombination (HR) and suppressing single-strand annealing (SSA). Consistent with this observation, cells depleted of FKBP25 form fewer Rad51 repair foci in response to etoposide and ionizing radiation, and they are reliant on the SSA repair factor Rad52 for viability. We find that FKBP25's catalytic activity is required for promoting DNA repair, which is the first description of a biological function for this enzyme activity. Consistent with the importance of the FKBP catalytic site in HR, rapamycin treatment also impairs homologous recombination, and this effect is at least in part independent of mTor. Taken together these results identify FKBP25 as a component of the DNA DSB repair pathway.

Keywords: DNA repair; FKBP; homologous recombination; olaparib; prolyl isomerase; prolyl isomérase; recombinaison homologue; réparation d’ADN.

Fig. 1.

FKBP25 promotes homologous recombination and suppresses single-strand annealing double strand break (DSB) repair pathways. (A–D) Flow cytometry reporter assay measuring DSB repair pathway utilization in FKBP25 knockdown U2OS cells containing stably integrated reporters for (A) Homologous Recombination, (B) Non-Homologous End Joining, (C) single-strand annealing, and (D) Alternative End Joining. Error bars represent the standard error of 4 independent replicates. *, P < 0.05; ***, P < 0.001.

Fig. 2.

FKBP25 promotes Rad51 foci formation in response to DNA damage. (A) FKBP25 expression levels relative to GAPDH in U2OS shRNA knockdown cells. (B) Rad51 foci formation in response to etoposide. shRNA knockdown cells treated with 100 μmol/L washed and incubated for 2 h; 20 cells counted per sample. Also shown, quantification of cells with greater or less than 20 Rad51 foci per cell (C) Rad51 requirement in response to ionizing radiation. Cells treated with 5 Gy of radiation and incubated for 2 h; 60 cells counted per condition. Also shown, quantification of cells with greater or less than 20 Rad51 foci per cell. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Fig. 3.

Cells deficient for FKBP25 and the single-strand annealing (SSA) repair factor Rad52 exhibit synthetic loss of viability. (A) Cell counting assay quantifying proliferation in U2OS cells stably expressing shRNA targeting green fluorescent protein (GFP) or FKBP25 and transfected with pooled siRNA targeting Rad52 or a non-targeting control. Error bars represent the standard error of 4 replicates in 2 independent experiments. (B) MTT proliferation assay of U2OS cells transfected with different combinations of siRNA, targeting FKBP25, Rad52, or a non-targeting control. Assay performed 72 h post-transfection. Data points shown are multiple measurements taken from 2 independent experiments. Error bars represent the standard error of 4 measurements across 4 independent transfections. **, P < 0.01; ***, P < 0.001.

Fig. 4.

FKBP25 is displaced from laser microirradiation-induced DNA double-strand breaks. Quantification of fluorescent intensity of FKBP25-NLS-GFP after laser-induced damage (top). Images depicting the site of exclusion from laser-induced damage are shown below. The laser microirradiated area is indicated with an arrow.

Fig. 5.

FKBP25’s catalytic activity is required for homologous recombination (HR). (A) Western blot analysis of the U2OS DR-GFP homologous recombination reporter cells transfected with I-SceI and either an empty vector control, FKBP25(Wt), or FKBP25(Y198F) expression vectors. (B) Quantification of GFP U2OS DR-GFP reporter cells transfected as in A by flow cytometry. Error bars represent standard error of 3 independent transfections. *, P < 0.05.

Fig. 6.

Inhibition of FKBPs impairs homologous recombination independently of mTOR. (A) A schematic presenting the strategy for chemical inhibition of FKBP25 by rapamycin without inhibiting mTOR. mTOR activity is restored by competition with FK506, which has a similar affinity for FKBP12 as rapamycin. [The following Protein Databank (PDB) entries were used in the generation of this figure; PDB ID 3FAP (Liang et al. 1999) and PDB ID 2MPH (Prakash et al. 2016)] (B) MTT proliferation assay as a proxy for the restoration of mTOR activity measuring proliferation. Cells were treated with either a dimethyl sulfoxide (DMSO) control, 10 nmol/L rapamycin, or 10 nmol/L Torin1 in the absence or presence of 2 μmol/L FK506. Error bars represent the standard error of 4 measurements across 4 independent experiments. (C) MTT proliferation assay of cells treated with increasing doses of the Parp-inhibitor olaparib in combination with 10 nmol/L rapamycin or 10 nmol/L rapamycin and 2 μmol/L FK506. Error bars represent the standard error of 4 measurements across 4 independent experiments. (D) MTT proliferation assay, cells treated as in (E). Error bars represent the standard error of 4 measurements from 2 independent experiments. (E) Flow cytometry reporter assay measuring the homolous recombinant double strand break repair pathway utilization in cells treated with DMSO control; 10 nmol/L rapamycin, 10 nmol/L Torin 1, and 2 μmol/L FK506; or 10 nmol/L rapamycin and 2 μmol/L FK506. Error bars represent standard error of 4 independent experiments. Significance relative to the DMSO control treated cells is shown above each bar. *, P < 0.05; **, P < 0.01.