ATRX and RECQ5 define distinct homologous recombination subpathways

Abstract

Homologous recombination (HR) is an important DNA double-strand break (DSB) repair pathway that copies sequence information lost at the break site from an undamaged homologous template. This involves the formation of a recombination structure that is processed to restore the original sequence but also harbors the potential for crossover (CO) formation between the participating molecules. Synthesis-dependent strand annealing (SDSA) is an HR subpathway that prevents CO formation and is thought to predominate in mammalian cells. The chromatin remodeler ATRX promotes an alternative HR subpathway that has the potential to form COs. Here, we show that ATRX-dependent HR outcompetes RECQ5-dependent SDSA for the repair of most two-ended DSBs in human cells and leads to the frequent formation of COs, assessed by measuring sister chromatid exchanges (SCEs). We provide evidence that subpathway choice is dependent on interaction of both ATRX and RECQ5 with proliferating cell nuclear antigen. We also show that the subpathway usage varies among different cancer cell lines and compare it to untransformed cells. We further observe HR intermediates arising as ionizing radiation (IR)-induced ultra-fine bridges only in cells expressing ATRX and lacking MUS81 and GEN1. Consistently, damage-induced MUS81 recruitment is only observed in ATRX-expressing cells. Cells lacking BLM show similar MUS81 recruitment and IR-induced SCE formation as control cells. Collectively, these results suggest that the ATRX pathway involves the formation of HR intermediates whose processing is entirely dependent on MUS81 and GEN1 and independent of BLM. We propose that the predominant ATRX-dependent HR subpathway forms joint molecules distinct from classical Holliday junctions.

Keywords: DNA repair synthesis; Holliday junctions; crossovers; sister chromatid exchanges; synthesis-dependent strand annealing.

Fig. 1.

ATRX and RECQ5 define distinct subpathways of HR. (A) U2OS^ATRX cells, with and without doxycycline-induced ATRX expression, were transfected with siCtrl or siRECQ5 and treated with RAD51 inhibitor (RAD51i) prior to IR and throughout repair incubation. γH2AX foci were enumerated in EdU-negative G2 cells. Spontaneous foci (four to six) were subtracted. Knockdown and ATRX expression were confirmed by immunoblotting. Representative images of γH2AX foci and ATRX expression are shown. (B) U2OS cells were transfected with siCtrl or siRECQ5-2 and GFP, GFP-RECQ5-WT, GFP-RECQ5-ATP, or GFP-RECQ5-PIP, irradiated and γH2AX foci were enumerated in GFP-positive, EdU-negative G2 cells. Knockdown of the endogenous RECQ5 and GFP-RECQ5 expression levels were confirmed by immunoblotting (SI Appendix, Fig. S2A). Spontaneous foci (four to six) were subtracted. Representative images of γH2AX foci in GFP-positive cells are shown. (C) U2OS cells were transfected with GFP, GFP-ATRX-WT, GFP-ATRX-ATP, or GFP-ATRX-PIP, irradiated and γH2AX foci were enumerated in GFP-positive, EdU-negative G2 cells. Spontaneous foci (four to six) were subtracted. All data show mean ± SEM (n = 3). Results from individual experiments, each derived from 40 cells, are indicated. *P < 0.05; **P < 0.01; ***P < 0.001; ns: not significant (two-tailed t test).

Fig. 2.

ATRX and RECQ5 differentially affect long tract gene conversion and SCE formation. (A) U2OS^ATRX cells, with and without doxycycline-induced ATRX expression and transfected with siCtrl or siRECQ5, were labeled with EdU, irradiated, and then incubated with BrdU for 8 h. BrdU foci representing DNA repair synthesis were enumerated in EdU-negative G2 cells. Spontaneous foci (fewer than one) were subtracted. (B) Schematic diagram of the LTGC/STGC reporter system. U2OS cells carry a 5′ truncated GFP cassette followed by two incorrectly oriented exons (exon B placed 5′ to exon A) of the Blasticidin resistance (BsdR) gene, and a GFP cassette that is disrupted by the recognition sequence of the I-SceI endonuclease. To obtain a functional GFP protein, the break has to be repaired by HR using the truncated GFP cassette as a template. STGC events of fewer than 1 kbp result in a functional GFP gene but fail to correctly orient the exons of the BsdR gene, and the cells are thus sensitive to Blasticidin. LTGC events of >1 kbp restore the GFP sequence and additionally place exon B of the BsdR gene 3′ to exon A, reinstating correct orientation and conferring Blasticidin resistance. Thus, LTGC events lead to GFP⁺, BsdR⁺ cells [image modified from Nagaraju et al. (45)]. For the analysis, GFP⁺ cells (representing both STGC and LTGC events) and BsdR⁺ cells (LTGC events) are enumerated. (C) U2OS STGC/LTGC reporter cells were transfected with siCtrl or siRECQ5 and pUC19 or myc-ATRX and 40 h later transfected with I-SceI plasmids. Cells were assessed for GFP expression and Blasticidin resistance and numbers were plotted as a fraction of all cells. Control experiments showed that the fraction of S/G2-phase cells varied by less than 5% between siRECQ5-treated, ATRX-expressing, and control cells, excluding that changes in cell cycle distributions substantially influence the results. Knockdown was confirmed by immunoblotting and myc-ATRX expression efficiency was previously assessed (26). The significance indication above the white bar compares the LTGC fraction of pUC19 to the ATRX-expressing siCtrl-treated cells; indications above the black bars compare STGC fractions upon RECQ5 depletion to the respective siCtrl-treated cells. (D) U2OS^ATRX cells, with and without doxycycline-induced ATRX expression, were transfected with siCtrl or siRECQ5 and incubated with BrdU for 48 h. Cells were then irradiated with 2 Gy, collected after 16 h, and processed to obtain mitotic spreads. SCEs per spread were quantified and normalized to 70 chromosomes. Representative images of chromosome spreads of nonirradiated and irradiated siCtrl-treated cells with and without ATRX expression are shown; red arrows in the magnifications show individual SCE events. Data in A and C show mean ± SEM (n = 3). Results from individual experiments, each derived from 40 cells in A, are indicated. Individual SCE data are shown and red horizontal lines indicate the mean; 70 to 120 spreads and >4,000 chromosomes per condition were analyzed from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ns: not significant (two-tailed t test); NIR: nonirradiated.

Fig. 3.

HR subpathway usage in ATRX-proficient cancer and normal cells. (A) HeLa WT and ATRX KO cells were transfected with siCtrl or siRECQ5 and treated with RAD51i prior to IR and throughout repair incubation. γH2AX foci were enumerated in EdU-negative G2 cells. Spontaneous foci (three to four) were subtracted. Knockdown was confirmed by immunoblotting. Data for siCtrl were extracted from supplemental appendix, figure S3A in ref. as they were part of the same experiment with siRECQ5. (B) HeLa WT and ATRX KO cells were transfected with siCtrl or siRECQ5 and incubated with BrdU for 48 h. Cells were then irradiated with 2 Gy, collected after 8 h, and processed to obtain mitotic spreads. SCEs per spread were quantified and normalized to 70 chromosomes. (C) 82-6 cells were transfected with siCtrl, siRECQ5, and/or siATRX and treated with RAD51i prior to IR and throughout repair incubation. γH2AX foci were enumerated in EdU-negative G2 cells. Spontaneous foci (one to two) were subtracted. Knockdown was confirmed by immunoblotting. (D) 82-6 cells were transfected with siCtrl, siRECQ5, and/or siATRX and incubated with BrdU for 48 h. Cells were then irradiated with 2 Gy, collected after 12 h, and processed to obtain mitotic spreads. SCEs per spread were quantified and normalized to 46 chromosomes. Foci data show mean ± SEM (n = 3). Results from individual experiments, each derived from 40 cells, are indicated. Individual SCE data are shown and red horizontal lines indicate the mean; 70 to 120 spreads and >4,000 chromosomes per condition were analyzed from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ns: not significant (two-tailed t test).

Fig. 4.

ATRX-dependent HR leads to the formation of IR-induced MUS81-dependent HR intermediates. (A) HeLa and U2OS cells were transfected twice on consecutive days with siCtrl or siMUS81+siGEN1, irradiated, and RPA- and/or BLM-positive UFBs were enumerated in EdU-negative late anaphase cells. Representative images of UFBs in MUS81- and GEN1-depleted HeLa and U2OS cells are shown. Knockdown was confirmed by immunoblotting. (B) HeLa and U2OS cells were irradiated with 2 Gy, prophase cells positive for the mitotic marker pH3 and negative for EdU were selected, and Z-stack images were captured and analyzed for MUS81 foci numbers and colocalization with γH2AX foci. All data show mean ± SEM (n = 3) and results from individual experiments, each derived from 40 (A) or 20 (B) cells, are indicated. **P < 0.01; ***P < 0.001; ns: not significant (two-tailed t test); NIR: nonirradiated.

Fig. 5.

Processing of ATRX-dependent HR intermediates is BLM independent. (A) HeLa and U2OS cells were transfected with siCtrl or siBLM, irradiated, and γH2AX (Left) and RAD51 (Right) foci were enumerated in EdU-negative G2 cells. Spontaneous foci (four to six γH2AX and fewer than one RAD51) were subtracted. Knockdown was confirmed by immunoblotting. (B) HeLa and U2OS cells were transfected with siCtrl or siBLM, irradiated with 2 Gy, and MUS81 foci were enumerated in pH3-positive, EdU-negative prophase cells. (C) HeLa cells were transfected with siCtrl or siBLM and incubated with BrdU for 48 h. Cells were then irradiated with 2 Gy, collected after 8 h, and processed to obtain mitotic spreads. SCEs per spread were quantified and normalized to 70 chromosomes. Bar graph shows the IR-induced SCE numbers (total SCEs after background subtraction). Representative images of chromosome spreads from unirradiated siCtrl- and siBLM-treated cells are shown on the right; red arrows in the magnifications show individual SCE events. Individual SCE data are shown and red horizontal lines indicate the mean; 70 to 120 spreads and >4,000 chromosomes per condition were analyzed from three independent experiments. Foci and IR-induced SCE data show mean ± SEM (n = 3) and results from individual experiments, each derived from 40 (A and C) or 20 (B) cells, are indicated. *P < 0.05; **P < 0.01; ***P < 0.001; ns: not significant (two-tailed t test); NIR: nonirradiated.

Fig. 6.

Model for the interplay between ATRX and RECQ5 to regulate distinct subpathways of HR. Repair of two-ended DSBs by ATRX-mediated HR outcompetes RECQ5, possibly through PCNA binding, thereby suppressing SDSA. Upon the completion of DNA repair synthesis, the resulting JM is not subject to dissolution by BLM and is channelled into the resolution pathway by MUS81 and GEN1, leading to equal probability of CO and non-CO products.