The DDN catalytic motif is required for Metnase functions in non-homologous end joining (NHEJ) repair and replication restart

Abstract

Metnase (or SETMAR) arose from a chimeric fusion of the Hsmar1 transposase downstream of a protein methylase in anthropoid primates. Although the Metnase transposase domain has been largely conserved, its catalytic motif (DDN) differs from the DDD motif of related transposases, which may be important for its role as a DNA repair factor and its enzymatic activities. Here, we show that substitution of DDN(610) with either DDD(610) or DDE(610) significantly reduced in vivo functions of Metnase in NHEJ repair and accelerated restart of replication forks. We next tested whether the DDD or DDE mutants cleave single-strand extensions and flaps in partial duplex DNA and pseudo-Tyr structures that mimic stalled replication forks. Neither substrate is cleaved by the DDD or DDE mutant, under the conditions where wild-type Metnase effectively cleaves ssDNA overhangs. We then characterized the ssDNA-binding activity of the Metnase transposase domain and found that the catalytic domain binds ssDNA but not dsDNA, whereas dsDNA binding activity resides in the helix-turn-helix DNA binding domain. Substitution of Asn-610 with either Asp or Glu within the transposase domain significantly reduces ssDNA binding activity. Collectively, our results suggest that a single mutation DDN(610) → DDD(610), which restores the ancestral catalytic site, results in loss of function in Metnase.

Keywords: DNA Binding Protein; DNA Damage; DNA Enzymes; DNA Repair; DNA Replication.

FIGURE 1.

The active site of the Metnase transposase catalytic domain (Protein Data Bank code 3K9J) is shown superimposed on the same region from Mos1 (Protein Data Bank 2F7T). Catalytically important residues in Metnase include Asp-483, Asp-575, and Asn-610 shown in a stick model with carbons (green), oxygens (red), nitrogens (blue), and a gray ribbon for the surrounding protein structure. In this structure, Ca²⁺ (orange sphere) is coordinated by Asp-483. Mos1 residues are indicated with an asterisk and include Asp-156, Asp-249, and Asp-284, shown in a stick model with carbon (blue) and oxygen (red) and the surrounding protein structure shown in a light blue ribbon. Mg²⁺ (yellow sphere) is coordinated by Asp-156 and Asp-249 in this structure.

FIGURE 2.

The catalytic DDN⁶¹⁰ motif is critical for role of Metnase in DSB repair. A, siRNA knockdown of Metnase analyzed by RT-PCR. B, representative images of HEK293 cells mock-transfected (top) or transfected with control siRNA (middle) or Metnase siRNA (bottom). Cells were treated with 2 mm HU for 18 h, released into fresh media at indicated times, stained with DAPI (blue) and antibodies to γ-H2AX (green), and imaged by confocal microscopy. C, percentage of γ-H2AX positive cells treated with either control- or Metnase-specific siRNA. Values are averages ± S.D. for two determinations. D, Western blot analysis of FLAG-tagged WT, N610D, and N610E Metnase stably expressed in HEK293 cells using an anti-FLAG monoclonal antibody. Ku80 was used as a loading control. E, representative confocal microscope images of HEK293 transfected with pFlagCMV4 vector and HEK293T cells overexpressing WT or mutant Metnase following mock or 18 h treatment with 2 mm HU, released into fresh medium for indicated times, cytospun, stained with DAPI (blue) and antibodies to γ-H2AX (green), and imaged by confocal microscopy. F, quantitation of E. Plotted are average percentages (± S.D.) of γ-H2AX-positive cells. An average of >200 cells were counted per slide, six slides per experiment. G, in vitro DNA end joining in cell-free extracts from HEK293 stably expressing WT or N610D Metnase. Cell extracts were incubated with linearized pBS DNA (1.0 μg), 1 mm MgCl₂, and 1 mm ATP for 60 min, DNA was isolated and transformed into E. coli for colony counts. Values are averages (±S.D.) of three or more DNA end-joining assays. *, p < 0.05; **, p < 0.01, t tests. H, the catalytic motif DDN⁶¹⁰ is crucial for the role of Metnase in DNA end joining coupled to genomic integration. HEK293 cells stably transfected with pFlag2 (vector) or pFlag2 expressing WT, N610D, or N610E Metnase were transfected with 2 or 4 μg) of KpnI-linearized pRNA/U6.Hygro plasmid, Hyg^r colonies were scored 14 days later to assess DNA integration. Values are averages (± S.D.) for six determinations. **, p < 0.01, t tests.

FIGURE 3.

The Metnase catalytic DDN⁶¹⁰ motif has a critical role in replication fork restart following HU treatment. A, dual labeling protocol for DNA fiber analysis. Cells were pulse-labeled with IdU (red) for 20 min (20′), treated with 0 or 5 mm HU for 60 min, and labeled with CldU (green) for the indicated times. B, representative confocal microscope images of replication tracks from HEK293 cells treated with either control- (top) or Metnase-specific siRNA (bottom). Cells were treated or mock treated with HU and pulse-labeled with CldU for 15 min. C, as in B but CldU pulse labeled for 10, 15, 20, or 30 min. Arrows indicate short green patches representing early fork restart. D, representative images of replication tracks from HEK293 cells stably transfected with empty vector control (top) or with WT or N610D mutant Metnase vector (middle and bottom) and pulse-labeled with CldU for 15 min following 60 min HU treatment. E–G, average percentages (± S.D.) of stopped (red only), restarted (red plus green), and new forks (green only) for three independent determinations. *, p < 0.05; **, p < 0.01, t tests.

FIGURE 4.

Metnase cleavage of ss-overhangs requires Mg²⁺ and the DDN⁶¹⁰ motif. A, silver staining of purified WT and mutant Metnase (50 ng) following 10% SDS-PAGE. B, ss-overhang cleavage activity of Metnase in the presence of different metal ion. Reaction mixtures (20 μl) containing 50 fmol of 5′-³²P-labeled 5′-flap DNA and 100 ng of WT-Metnase in the presence of 2 and 5 mm of indicated metal ion were incubated for 60 min at 37 °C, and cleavage products were analyzed by 12% PAGE containing 8 m urea. DNA size makers are indicated on the left. C, E, and F, DNA cleavage of 5′-overhang partial duplex DNA (C), 5′-flap DNA (E), and a fully replicated fork DNA (F) by WT Metnase, N610D, or N610E mutant Metnase (50 and 100 ng). D, Metnase DDD and DDE mutants show enhanced end cleavage activity with TIR DNA with either 3′- or 5′-ss-overhang. Reaction mixtures (20 μl) containing 50 fmol of 5′-³²P-labeled TIR DNA were incubated with 50 and 100 ng of either WT or mutant Metnase in the presence of 2 mm MgCl₂. After incubation for 60 min at 37 °C, cleavage products were analyzed by 12% PAGE containing 8 m urea. DNA sizes are indicated on the left.

FIGURE 5.

Binding curves of Metnase fragments to different DNA probes as measured by fluorescence anisotropy. A, Metnase transposase domain (aa 329–671) with 5′ 6-nt dsDNA (25/31-mer), K_d of 11.9 ± 2.5 nm. B, Metnase transposase domain (aa 329–671) and the catalytic domain (aa 433–671) with ssDNA (25 nt), K_d of 277 ± 26 nm and 295 ± 47 nm, respectively. The equilibrium binding data were analyzed by nonlinear curve fitting using the one site saturation ligand binding equation (SigmaPlot, version 11.2). The K_d values are presented as K_d ± S.E. S.E. represents the error calculated for the determination of the K_d value based on the fit of the data. At least two independent assays were done for each sample, and duplicate measurements were averaged for each concentration of protein used. C, WT-Metnase and DDD/DDE mutant bind TIR DNA. Gel mobility shift analysis of WT and mutant Metnase (0.05 or 0.1 μg) incubated with 5′-³²P-labeled TIR DNA (200 fmol) resolved on a 0.8% agarose gel.