Barrier-to-autointegration factor 1 (Banf1) regulates poly [ADP-ribose] polymerase 1 (PARP1) activity following oxidative DNA damage

Abstract

The DNA repair capacity of human cells declines with age, in a process that is not clearly understood. Mutation of the nuclear envelope protein barrier-to-autointegration factor 1 (Banf1) has previously been shown to cause a human progeroid disorder, Néstor-Guillermo progeria syndrome (NGPS). The underlying links between Banf1, DNA repair and the ageing process are unknown. Here, we report that Banf1 controls the DNA damage response to oxidative stress via regulation of poly [ADP-ribose] polymerase 1 (PARP1). Specifically, oxidative lesions promote direct binding of Banf1 to PARP1, a critical NAD⁺-dependent DNA repair protein, leading to inhibition of PARP1 auto-ADP-ribosylation and defective repair of oxidative lesions, in cells with increased Banf1. Consistent with this, cells from patients with NGPS have defective PARP1 activity and impaired repair of oxidative lesions. These data support a model whereby Banf1 is crucial to reset oxidative-stress-induced PARP1 activity. Together, these data offer insight into Banf1-regulated, PARP1-directed repair of oxidative lesions.

Fig. 1

Banf1 responds to oxidative stress. a Banf1 relocalises from the nuclear envelope following oxidative stress induced by 200 μM H₂O₂ in U2OS cells. Representative cells are shown. b The nuclear intensity of Banf1 in U2OS cells treated as in (a), were analysed via an InCell Analyser 2200. One-way ANOVA was used for statistical analysis. c Banf1 relocalisation following hydrogen peroxide (H₂O₂), camptothecin (CPT), or potassium bromate (KBrO₃) at the indicated times post compound removal. Representative cells stained with the indicated antibodies are shown. d Nuclear envelope localisation (from (c)) was assessed using a delta vision PDV microscope. Immunofluorescence scale bars represents 10 μm. Histogram data shown represent the mean and S.D. of three independent experiments. ANOVA **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.

Fig. 2

Banf1 binds to PARP1 following oxidative stress. a, b Banf1 and PARP1 are in a complex; immunoprecipitations from HEK293T cells expressing Flag, Flag-Banf1 or Flag-PARP1 using Flag antibodies. Immunoprecipitates were immunoblotted with the indicated antibodies. c The Banf1:PARP1 interaction after H₂O₂; immunoprecipitations from HEK293T cells ectopically expressing Flag or Flag-Banf1 1 h after H₂O₂ removal. Immunoprecipitates were immunoblotted with the indicated antibodies. d Banf1 and PARP1 directly interact. The indicated purified proteins (±NAD⁺/DNA/olaparib) were incubated together before immunoprecipitation with PARP1 antibodies and immunoblotting with the indicated antibodies. e Banf1 and PARP colocalise following oxidative stress induced by H₂O₂ in U2OS cells. Representative cells fixed at the indicated times following removal of H₂O₂ and stained with the indicated antibodies are shown, the colocalisation was analysed using ImageJ. The scale bar represents 10 μm. Immunoblots are representative of three independent experiments. Source data are provided as a Source Data file.

Fig. 3

Banf1 regulates PARP1 activity and repair of oxidative DNA damage. a Auto-poly-ADP-ribosylation of PARP1 in U2OS cells depleted of Banf1 following H₂O₂. The PAR bands were analysed via densitometry and normalised to γ-Tubulin. Two-way ANOVA was used for statistical analysis. b Auto-poly-ADP-ribose of PARP1 in U2OS expressing ectopic Flag-Banf1 following H₂O₂. The PAR bands were analysed via densitometry and normalised to γ-Tubulin. Two-way ANOVA was used for statistical analysis. c Inhibition of poly-ADP-ribose activity of PARP1 purified from HEK293T cells expressing ectopic Flag-Banf1 on an immobilised histone substrate following H₂O₂. Data shown represent the mean and S.D. of two independent experiments. Paired t-test was used for statistical analysis. d In vitro inhibition of PARP1 poly-ADP-ribose activity on histones by purified Banf1. e Alkaline comet assay showing the relative olive tail moment in Banf1-deficient and control U2OS cells. f Banf1 inhibits repair of oxidative DNA damage. Alkaline comet assay showing the relative olive tail moment in U2OS control cells and cells ectopically expressing Flag-Banf1. Paired t test was used for statistical analysis. Histogram data shown in f represent the mean and S.D. of four independent experiments immunoblots are representative of three independent experiments. Unless otherwise stated, histogram data shown represent the mean and S.D. of three independent experiments. ANOVA was used for statistical analysis. *P < 0.05, **P < 0.01. Source data are provided as a Source Data file.

Fig. 4

The N-terminal of Banf1 binds to the NAD-binding domain of PARP1. a Structural superimposition of catalytic domains of chicken (blue colour) and human (golden colour) PARP1. NAD⁺ analogue is shown as ball and sticks and the conserved binding site residues shown as sticks. The loop consisting of residues 906–911 and 883–893 of PARP1 are highlighted in dark green and orange, respectively. b 3D structure of the most representative catalytic domain of PARP1–Banf1 obtained from Cluspro docking server. The Banf1 dimer is shown in red and green ribbons. The N-terminal of Banf1 monomer occupies the NAD⁺ binding site hence inhibiting NAD⁺ interaction. The interface residues and interactions at the PARP1–Banf1 interface are shown in the box where chain A and C represents residues from PARP1 and Banf1, respectively. The loop consisting of residues 906–911 of PARP1 are highlighted in dark green. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; E, Glu, D, Asp; N, Asn. c, d Interactions of Banf1 with Flag-PARP1 mutants. Flag immunoprecipitations from HEK293T cells ectopically expressing the indicated Flag-PARP1 proteins 1 h following H₂O₂ removal. e Interactions of PARP1 with Flag-Banf1 mutants. Flag immunoprecipitations from HEK293T cells ectopically expressing the indicated Flag-Banf1 proteins 1 h following H₂O₂ removal. f Analysis of the relative Banf1 WT vs. D9A binding to PARP1 via immunoprecipitation. g Analysis of the relative auto-poly-ADP-ribosylation of PARP1 in cells expressing WT or D9A Banf1. h In vitro inhibition of PARP1 binding NAD⁺ in the presence of purified Banf1. Histogram data shown in h, represent the mean and S.D. of four independent experiments. Immunoblots are representative of three independent experiments. Unless otherwise stated, histogram data shown represent the mean and S.D. of three independent experiments. Unpaired t test was used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.

Fig. 5

Banf1 A12T inhibits repair of oxidative lesions in NGPS patient cells. a Interactions of PARP1 with Flag-Banf1 mutants. Flag immunoprecipitations from HEK293T cells ectopically expressing the Flag-Banf1 WT or A12T proteins following H₂O₂. b Purified Banf1 WT or A12T proteins were incubated with PARP1, immunopreciptated with PARP1 antibodies and immunoblotted with the indicated antibodies. c Inhibition of auto-poly-ADP-ribose activity of PARP1 in U2OS expressing ectopic Flag-Banf1 WT or A12T following H₂O₂. d The PAR bands in (c), were analysed via densitometry and normalised to γ-Tubulin. Histogram data shown in (d), represent the mean and S.D. of three independent experiments. e In vitro inhibition of PARP1 poly-ADP-ribose activity on histones by purified Banf1 WT or A12T. f The PAR bands on (e), were analysed via densitometry. g Banf1 inhibits repair of oxidative DNA damage. Alkaline comet assay showing the relative olive tail moment in control cells and cells ectopically expressing Flag-Banf1 WT or A12T following H₂O₂ treatment and recovery. Paired t test was used for statistical analysis. h Inhibition of Poly-ADP-ribose activity of PARP1 purified from NGPS patient cells on an immobilised histone substrate HEK293T following H₂O₂. i NGPS patient cells exhibit defective repair of oxidative DNA damage. Alkaline comet assay showing the relative olive tail moment in control cells and NGPS cells after H₂O₂ treatment and recovery. Paired t test was used for statistical analysis. Immunoblots are representative of n = 3 independent experiments. Unless otherwise stated, histogram data shown represent the mean and S.D. of n = 4 independent experiments and statistical significance was defined via ANOVA. *P < 0.05, **P < 0.01. Source data are provided as a Source Data file.