AUNIP/C1orf135 directs DNA double-strand breaks towards the homologous recombination repair pathway

Abstract

DNA double-strand breaks (DSBs) are mainly repaired by either homologous recombination (HR) or non-homologous end-joining (NHEJ). Here, we identify AUNIP/C1orf135, a largely uncharacterized protein, as a key determinant of DSB repair pathway choice. AUNIP physically interacts with CtIP and is required for efficient CtIP accumulation at DSBs. AUNIP possesses intrinsic DNA-binding ability with a strong preference for DNA substrates that mimic structures generated at stalled replication forks. This ability to bind DNA is necessary for the recruitment of AUNIP and its binding partner CtIP to DSBs, which in turn drives CtIP-dependent DNA-end resection and HR repair. Accordingly, loss of AUNIP or ablation of its ability to bind to DNA results in cell hypersensitivity toward a variety of DSB-inducing agents, particularly those that induce replication-associated DSBs. Our findings provide new insights into the molecular mechanism by which DSBs are recognized and channeled to the HR repair pathway.DNA double strand breaks can be repaired by homology-independent or homology-directed mechanisms. The choice between these pathways is a key event for genomic stability maintenance. Here the authors identify and characterize AUNIP, as a factor involved in tilting the balance towards homology repair.

Fig. 1

Identification of AUNIP as a CtIP-associated protein. a, c CtIP (a) or AUNIP (c) protein complexes separated by SDS-PAGE were stained with Coomassie blue. b, d Proteins identified by mass spectrometric analyses of CtIP or AUNIP protein complexes are listed. Bait proteins are indicated in bold letters. e AUNIP interacts with CtIP and the MRN complex. HeLa cell lysates treated with Benzonase were precipitated with anti-AUNIP or anti-CtIP antibodies and were analyzed by immunoblotting with indicated antibodies. f AUNIP interacts with CtIP via its C-terminus. HEK293T cells transfected with indicated plasmids were lysed with NETN buffer 24 h post transfection. Cell lysates were then incubated with S-protein beads and immunoprecipitated proteins were analyzed by immunoblotting experiments using indicated antibodies. g Direct in vitro binding between recombinant GST-AUNIP and MBP-CtIP purified from E. coli. GST served as a negative control for CtIP binding. Top: CtIP was detected by immunoblotting experiment. Bottom: purified proteins were visualized by Coomassie staining

Fig. 2

AUNIP accumulates at perturbed replication forks and at sites of DNA damage. a Endogenous AUNIP accumulates at perturbed replication forks. AUNIP-SFB knock-in HeLa cells were either mock treated or treated with 1 μM CPT for 6 h before they were processed for indirect immunofluorescent analysis for AUNIP-SFB (α-Flag) and RPA2 (α-RPA2). b GFP-tagged AUNIP is recruited to laser-induced DNA damage sites. U2OS cells transfected with GFP-tagged AUNIP were laser micro-irradiated. After 10 min, cells were stained with anti-RPA2 or anti-γH2AX antibody. c AUNIP-SFB knock-in HeLa cells were laser micro-irradiated. Ten minutes post irradiation, cells were processed for immunostaining experiments using anti-Flag and anti-RPA2/γH2AX antibodies. d Schematic representation of AUNIP mutants used in this study. e Amino acids 25–70 of AUNIP is required for its recruitment to sites of DNA damage. Cells transfected with GFP-tagged wild-type AUNIP or its mutants were laser micro-irradiated. Cells were processed for immunostaining experiments using anti-γH2AX antibody. f Amino acids 21–70 of AUNIP is sufficient for its recruitment to sites of DNA damage. Cells transfected with GFP-AUNIP-21–70 were laser micro-irradiated. Cells were labeled with anti-γH2AX antibody to visualize DNA damage tracks

Fig. 3

AUNIP promotes HR and inhibits NHEJ. a Schematic illustration of the GFP-based HR reporter assay. b AUNIP overexpression promotes HR. U2OS DR-GFP cells stably expressing Flag-tagged AUNIP were electroporated with pCBASce construct and were assayed for HR efficiency by monitoring GFP positivity 48 h post electroporation. Data represent mean ± SEM from three independent experiments. c Schematic illustration of the GFP-based NHEJ reporter assay. d AUNIP overexpression inhibits NHEJ. U2OS EJ5-GFP cells stably expressing Flag-tagged AUNIP were electroporated with pCBASce construct and were assayed for NHEJ efficiency by monitoring GFP positivity 48 h post electroporation. Data represent mean ± SEM from three independent experiments. e AUNIP knockout impairs HR. AUNIP-deficient U2OS DR-GFP cells stably expressing an empty vector (Vector), Flag-tagged wild-type AUNIP, the Δ25–70 mutant, or the 1–280 mutant were electroporated with pCBASce construct for 48 h before they were assayed for HR efficiency. Data represent mean ± SEM from three independent experiments. f The overall levels of a panel of key HR proteins are not markedly affected by AUNIP silencing. Whole-cell lysates from indicated cells were subjected to western blot analysis using indicated antibodies. Asterisks indicate nonspecific bands. g, h Knockout of AUNIP impairs CPT-induced RAD51 foci formation. AUNIP-deficient cells stably expressing an empty vector (Vector), Flag-tagged wild-type AUNIP, the Δ25–70 mutant, or the 1–280 mutant were treated with 1 μM CPT for 6 h before being processed for RAD51 immunofluorescence. Representative RAD51 foci are shown in g. Data represent mean ± SEM from three independent experiments (h)

Fig. 4

AUNIP stimulates DNA end resection. a–d Knockout of AUNIP impairs CPT-or X-ray-induced RPA2 foci formation. AUNIP-deficient HeLa cells were treated with 1  μM CPT or 10 Gy X-rays and were processed for RPA2 immunostaining experiment 1 h after. Representative RPA2 foci are shown in a, c. Data represent mean ± SEM from three independent experiments (b, d). e, f AUNIP does not affect HU-induced RPA2 foci formation. AUNIP-deficient HeLa cells were treated with 10 mM HU for 1 h before being processed for immunofluorescence studies using anti-RPA2 antibody. Representative RPA2 foci are shown in e. Data represent mean ± SEM from three independent experiments f. (g, h) Knockout of AUNIP impairs ssDNA formation. Wild-type or AUNIP-deficient U2OS cells were labeled with BrdU for 24 h, and were subsequently treated with 1 μM CPT for 1 h. Cells were then stained with anti-BrdU antibody under non-denaturing conditions. Representative BrdU foci are shown in g. Data represent mean ± SEM from three independent experiments (h). i Knockout of AUNIP impairs CPT-induced RPA2 phosphorylation. Cells were treated with 1  μM CPT for 1 h and were processed for western blot analysis. j–l The AUNIP mutants lacking the ability to either localize to DNA damage sites or to bind to CtIP failed to restore RPA2 phosphorylation and RPA2/BrdU foci formation in AUNIP-deficient cells. AUNIP-deficient cells stably expressing vector control (Vector), wild-type AUNIP, the Δ25–70 mutant, or the 1–280 mutant were treated with 1  μM CPT for 1 h. Cells were subsequently processed for western blot analysis (j) or for indirect immunofluorescence studies using anti-RPA2 (k) or anti-BrdU antibody (l)

Fig. 5

AUNIP promotes CtIP recruitment to DNA damage sites. a, b Kinetic studies of accumulation of GFP-tagged AUNIP, NBS1, or CtIP at laser-induced DNA damage sites. U2OS cells stably expressing GFP-AUNIP, NBS1-GFP, or GFP-CtIP were laser micro-irradiated and were monitored by live cell imaging (a). The intensity of fluorescence at the site of damage was quantified b. Data were derived from analysis of at least 20 cells in each experiment and are presented as mean ± SEM. c, d Knockout of AUNIP impairs CtIP recruitment to laser-induced DNA damage sites. AUNIP-deficient U2OS cells stably expressing empty vector (Vector), wild-type AUNIP, the Δ25–70 mutant, or the 1–280 mutant were infected with a lentiviral vector expressing GFP-tagged CtIP. After 48 h, cells were laser micro-irradiated and were monitored by live cell imaging (c). Data were derived from analysis of at least 20 cells in each experiment and are presented as mean ± SEM (d). e, f Knockout of AUNIP impairs CPT-induced CtIP foci formation. Cells infected with a lentiviral vector encoding GFP-tagged CtIP were treated with 1 μM CPT for 1 h and were processed for immunofluorescence studies. Representative CtIP foci are shown in e. Data represent mean ± SEM from three independent experiments (f). g–i Neither NBS1 nor CtIP is required for AUNIP damage recruitment. Cells transfected with indicated siRNAs were laser micro-irradiated and were monitored by live cell imaging (h). Data were derived from analysis of at least 20 cells in each experiment and are presented as mean ± SEM (i). Knockdown efficiency of NBS1/CtIP was confirmed by western blotting (g)

Fig. 6

DNA-binding activity of AUNIP. a SDS-PAGE profile of purified MBP-tagged wild-type and mutants of AUNIP. b–h DNA-binding activity of AUNIP. Biotin-labeled DNA substrates (20 nM each) were incubated with increasing amounts of purified MBP-AUNIP (0, 50, 100, 150, 200 nM). i Quantification of the results is shown in b–h. Data were derived from three independent experiments and are presented as mean ± SEM. j The region encompassing amino acids 25–70 of AUNIP is required for its DNA-binding activity. Biotin-labeled DNA substrates (20 nM) were incubated with increasing amounts of purified MBP-tagged AUNIP or its mutant (0, 100, 200 nM). k The region encompassing amino acids 21–70 of AUNIP is sufficient for DNA binding. Biotin-labeled DNA substrates (20 nM) were incubated with increasing amounts of purified MBP-AUNIP or its mutant (0, 100, 200 nM)

Fig. 7

AUNIP is required for genome stability and cellular resistance to DSB-inducing agents. a Wild-type or AUNIP-deficient HeLa cells were either mock treated or treated with 40 μM CPT for 12 h. Metaphase spreads were then prepared following standard procedures. Representative images are shown in a. Arrows indicate chromosome aberrations. Quantification of chromosomal aberrations from indicated cells is shown in b. Data represent the average for two independent experiments. At least 50 metaphases were counted in each experiment. c–f Sensitivity of wild-type cells or AUNIP-deficient HeLa cells to CPT, Topotecan, ABT-888, or IR was determined by a colony formation assay. Data represent means ± SEM from three independent experiments. g CPT sensitivity of wild-type or AUNIP-deficient cells that stably express empty vector (Vector), wild-type AUNIP, the Δ25–70 mutant, or the 1–280 mutant was determined by a colony formation assay. Data represent means ± SEM from three independent experiments. h Working model depicting a proposed role of AUNIP in DSB repair choice. Left panel: Noting that AUNIP displays low affinities for dsDNA ends, most of the two-ended DSBs are recognized and are repaired by the NHEJ machinery. Right panel: AUNIP enhances CtIP tethering at perturbed replication forks and directs replication-associated one-ended DSBs toward the HR repair pathway