Alteration of replication protein A binding mode on single-stranded DNA by NSMF potentiates RPA phosphorylation by ATR kinase

Abstract Replication protein A (RPA), a eukaryotic single-stranded DNA (ssDNA) binding protein, dynamically interacts with ssDNA in different binding modes and plays essential roles in DNA metabolism such as replication, repair, and recombination. RPA accumulation on ssDNA due to replication stress triggers the DNA damage response (DDR) by activating the ataxia telangiectasia and RAD3-related (ATR) kinase, which phosphorylates itself and downstream DDR factors, including RPA. We recently reported that the N-methyl-D-aspartate receptor synaptonuclear signaling and neuronal migration factor (NSMF), a neuronal protein associated with Kallmann syndrome, promotes RPA32 phosphorylation via ATR upon replication stress. However, how NSMF enhances ATR-mediated RPA32 phosphorylation remains elusive. Here, we demonstrate that NSMF colocalizes and physically interacts with RPA at DNA damage sites in vivo and in vitro. Using purified RPA and NSMF in biochemical and single-molecule assays, we find that NSMF selectively displaces RPA in the more weakly bound 8- and 20-nucleotide binding modes from ssDNA, allowing the retention of more stable RPA molecules in the 30-nt binding mode. The 30-nt binding mode of RPA enhances RPA32 phosphorylation by ATR, and phosphorylated RPA becomes stabilized on ssDNA. Our findings provide new mechanistic insight into how NSMF facilitates the role of RPA in the ATR pathway.


1-1. Cell culture
HeLa and human embryonic kidney (HEK) 293T cells were purchased from American Type Culture Collection (ATCC). The cell lines were maintained in Dulbecco-modified Eagle's medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Millipore) and 1% penicillin/streptomycin (Gibco) at 37°C under 5% v/v CO2. NSMF KO cell line was cultured as described previously (1). HeLa cells with stable Flag-NSMF-WT or D2 expression were obtained upon antibiotic selection with 3 μg/ml puromycin (InvivoGen) for 2 weeks. Clones were pooled into a single population to avoid clonal heterogeneity.

1-2. Laser microirradiation and imaging of cells
For laser microirradiation, HeLa cells were grown on 35 mm glass-bottom dishes (MatTek Corporation, Ashland, MA). Laser microirradiation was carried out using a Nikon A1 laser microdissection system equipped with a 37°C chamber and CO2 module (Nikon, Tokyo, Japan). Ultraviolet A laser with 355 nm wavelength was illuminated on selected regions at 100% power for 20 iterations to induce localized DNA damage. For GFP or mCherry-tagged proteins, timelapse images were acquired with 10 sec or 15 sec time intervals after laser microirradiation. The fluorescence intensity on each laser strip was recorded with NIS elements C software (Nikon) and analyzed with ImageJ software. The fluorescence values for >10 cells from three independent experiments were normalized to the original signal and plotted as fluorescence versus time using OriginPro (Origin Lab).
HEK293T cells were washed with ice-cold phosphate buffered saline (PBS) and then lysed in NETN buffer (0.5% nonidet P-40, 20 mM Tris-HCl [8.0], 50 mM NaCl, 50 mM NaF, 100 μM Na3VO4, 1 mM dithiothreitol (DTT), and 50 μg/ml phenylmethylsulfonyl fluoride (PMSF)), and 250 units/ml benzonase (M018H, Enzynomics) and 1 mM MgCl2 were added to each sample. The reactants were then incubated at 4°C for 45 min. Crude lysates were sonicated and cleared by ultracentrifugation at 15,800 g at 4°C for 10 min, and supernatants were incubated with protein A-agarose-conjugated antibody. The immunocomplexes were washed three times with NETN buffer and subjected to SDS-PAGE. Western blotting was performed using the antibodies indicated in the figures. Proteins were visualized using secondary horseradish peroxidase-conjugated antibodies (Enzo Life Sciences, New York, NY) and enhanced chemiluminescence reagent (ABC-3001, AbClon). Signals were detected using an automated imaging system (ChemiDoc™; Bio-Rad Laboratories).

Protein purification
All protein purification processes were performed at 4°C

3-2. Human wild-type RPA and DNA binding mutant RPA (DBM-RPA) purification
Human wild-type RPA with all three subunits (RPA70, RPA32, and RPA14) was purified as described in the previous literature (3). Briefly, the plasmid having all RPA subunits was kindly provided by Patrick Sung (UT Health San Antonio) and was transformed into E. coli BL21(DE3)pLysS strain (C6060-03, Thermo Fisher Scientific). 6 L cells in 2xLB media were grown at 37°C until OD600 ~ 0.6. The protein expression was induced by 1 mM IPTG and further incubated at 16°C overnight. Cells were harvested and resuspended in resuspension buffer (100 mM Tris-HCl [7.5], 600 mM KCl, 10 mM EDTA, 0.01% Igepal, 1 mM DTT, and 1 mM benzamidine). The cells were then lysed by sonication and spun down at 100,000 g at 4°C for 40 min. The clarified lysate was bound to 30 mL bed volume of Affi-gel blue column DNA binding mutant of RPA32 (DBM-RPA), where tryptophan 107 (W107) and phenylalanine 135 (F135) were both replaced by alanine, were also purified as the same procedure as the wild-type.

3-3. Human wild-type RPA-eGFP purification
Human wild-type RPA-eGFP (RPA-eGFP), in which eGFP is tagged at the C-terminus of RPA70, was purified as follows. The plasmid containing 6xHis-tagged RPA-eGFP was transformed into E. coli BL21(DE3)pLysS. 6 L cells in 2xLB media were grown at 37°C until OD600 ~ 0.6. The protein expression was induced by 1 mM IPTG and further incubated at 16°C overnight. Cells were then pelleted and resuspended in T-100 buffer with 1xprotease inhibitor (Halt, 78439, Thermo Fisher Scientific). Cells were lysed by sonication, and the lysate was clarified by centrifugation at 100,000 g for 1 hr at 4°C. The clarified lysate was loaded onto 30 mL bed volume of Affi-gel blue column (153-7302, Bio-Rad) that was equilibrated with T-100 buffer. The column was washed with T-100 buffer and further with T-800 buffer. Proteins were eluted by salt gradient from T-100 buffer to 2.5 M NaSCN buffer. The fractions containing RPA-eGFP were pooled and then diluted 10 times by Talon wash buffer (50 mM HEPES-NaOH [7.4], 250 mM NaCl, and 20 mM Imidazole) The protein was loaded onto 15 ml bed volume of Talon column (635670, TaKaRa) that was equilibrated with Talon wash buffer. After the column was washed with Talon wash buffer, RPA-eGFP was eluted with Talon elution buffer (Talon wash buffer supplemented with 500 mM imidazole). RPA-eGFP fractions were pooled and dialyzed against storage buffer (50 mM Tris-HCl [8.0], 40 mM NaCl, 0.5 mM EDTA, 1 mM DTT, and 10% glycerol). The final stock was stored at -80°C.

3-4. Human RPA subunit (RPA32 and RPA14) purification
Each subunit of human RPA (RPA32 and RPA14) was subcloned in pTXB1-derived plasmid having 6xHis at the N-terminus. Each plasmid was transformed into E. coli BL21(DE3) strain. 8 L cells were grown in LB media at 37°C until OD600 reached ~ 0.6, and then the protein expression was induced with 1 mM IPTG, and cells were further incubated at 16°C overnight. The cells were harvested and resuspended in T-100 buffer with 1xprotease inhibitor (Halt, 78439, Thermo Fisher Scientific). The cells were lysed using sonication, and the lysates were ultracentrifuged at 100,000 g for 70 min. The clarified lysates were loaded to 15 ml bed volume of HisPur™ Ni-NTA resin (88222, Thermo Fisher Scientific), which was pre-equilibrated with Ni-wash buffer (T-100 buffer supplemented with 20 mM imidazole). After Ni-NTA resin was washed with 5xcolumn volume (CV) of Ni-wash buffer, proteins were eluted with Ni-elution buffer (T-100 buffer supplemented with 500 mM imidazole). The fractions containing a RPA subunit were pooled and then diluted 10 times by T-buffer. Proteins were then purified by 1 ml Q column (HiTrap Q HP, 17115301, Cytiva) by salt gradient from T-100 buffer to T-1000 buffer. Fractions containing RPA32 or RPA14 were pooled and then dialyzed against 2 L storage buffer (50 mM Tris-HCl [8.0], 40 mM NaCl, 0.5 mM EDTA, 1 mM DTT, and 10% glycerol). Each purified RPA subunit was stored in -80°C after being snap-frozen by liquid N2.
The plasmid was transformed into E. coli BL21(DE3) strain. 8 L cells were grown in LB media at 37°C until OD600 reached ~ 0.6, and then the protein was expressed with 1 mM IPTG, and cells were further incubated at 16°C overnight. The cells were then harvested and resuspended in T-100 buffer with 1xprotease inhibitor (Halt, 78439, Thermo Fisher Scientific). The cells were lysed using sonication, and the lysates were ultracentrifuged at 100,000 g for 1 hr. The clarified lysates were loaded to 15 ml bed volume of HisPur™ Ni-NTA resin (88222, Thermo Fisher Scientific), which was pre-equilibrated with Ni-wash buffer (T-100 buffer with 20 mM imidazole). After Ni-NTA resin was washed with 5xCV of Ni-wash buffer, proteins were eluted with Ni-elution buffer (T-100 buffer with 500 mM imidazole). The fractions containing 7xHis RPA or 7xHis pmRPA were pooled and further purified by gel filtration with Superdex200 10/300 GL (17-5175-01, Cytiva) in T-100 buffer. Fractions containing 7xHis RPA or 7xHis pmRPA were pooled and then dialyzed against 2 L storage buffer (50 mM Tris-HCl [8.0], 40 mM NaCl, 0.5 mM EDTA, 1 mM DTT, and 10% glycerol). The purified protein was stored at -80°C after being snap-frozen by liquid N2.
7xHis phosphomimetic RPA that had either single S33D mutation or triple S4/8D and S33D mutations in RPA32 was purified by the same protocol as 7xHis wild-type RPA.

DNA preparation
Oligomers were all synthesized (Bioneer, South Korea). All DNA constructs were prepared by following the previous protocol (4). Oligomers were mixed at equi-molar ratio in 10 mM Tris-HCl [7.5] and 100 mM NaCl. For annealing, the mixtures were heated at 95°C for 10 min and slowly cooled to 23°C.

5-1-2. NSMF and various types of DNA substrates
All reactions were performed in reaction buffer (50 mM Tris-HCl [7.5], 50 mM NaCl, 1 mM DTT, and 0.01% Tween-20) at 23°C. 10 nM various types of Cy5-labeled DNA substrates, such as duplex, ssDNA, primer-template junction (PTJ), gap, bubble, D-loop, R-loop, or fork (Yshape), were incubated with NSMF at different concentrations in the reaction buffer for 30 min (Supplementary Table). The reactants were analyzed by running 1.5% agarose gel in 1xTBE buffer at 4°C, and the gel was imaged by Typhoon RGB (Cytiva). To estimate the dissociation equilibrium constant (Kd), we fitted the bound fraction graph with hyperbola function

5-2. Magnetic bead pulldown assay
300 nM of biotinylated 80-nt ssDNA was conjugated to 5 ul of streptavidin-coated magnetic beads (Dynabeads M-280, Invitrogen) in 20 mM Tris-HCl [8.0] and 100 mM NaCl at 23°C for 2 hrs. After unbound DNA was removed, 1.2 uM RPA was incubated with the ssDNAdecorated magnetic beads at 23°C for 50 min. Free RPA proteins were removed, and 20 nM NSMF or NSMF-D2 was incubated with the beads in reaction buffer for 30 min. Then the beads were pulled down and only supernatant was taken. The protein portion bound to ssDNA was eluted by boiling the beads at 95°C for 3 min. The supernatant and eluant were analyzed via western blot using an anti-FLAG antibody (A2220, Sigma) and anti-RPA32 antibody (A300-244A, Thermo Fisher Scientific).

5-3. Single-molecule photobleaching assay
The single-molecule photobleaching assay was conducted as described before (5). All reactions were performed at 23°C in reaction buffer. 0.3 nM biotinylated 14-, 30-, 60-, or 80-nt ssDNA was anchored on a streptavidin-coated slide surface (Supplementary Table 1). After unbound DNA was washed out, 0.75 nM RPA-eGFP was injected into the flowcell and incubated for 20 min. Free RPA proteins were washed out using 200 ul of reaction buffer containing 1xgloxy and 1.6% glucose three times, and 100 nM NSMF or NSMF-D2 mutant was incubated for 30 min. After residual NSMF was flushed out with reaction buffer containing 1xgloxy and 1.6% glucose, the fluorescence signal of RPA-eGFP was collected under the illumination of a 488 nm laser until almost all eGFP fluorescent puncta were photobleached. Fluorescence signals were imaged by NIS-element (Nikon). Images were analyzed by a customized software smCamera, which was kindly provided by Professor Kyung Suk Lee at Kongju National University. Each fluorescent punctum was fitted with a 2D Gaussian function, and then the fluorescence trajectories were extracted. The photobleaching steps were counted from each fluorescence intensity trajectory.

5-5. In vitro binding assay
Purified hRPA and eGFP-tagged NSMF WT or D-2 were used for in vitro binding assay. RPA and eGFP-NSMF WT or D-2 were incubated in NETN at 4°C for 1 hr and then anti-GFPcoupled protein G agarose (Pierce Protein G Agarose, 20399, Thermo Fisher Scientific) was added and additionally incubated at 4°C for 1 hr. The immunoprecipitates were washed three times with NETN and subjected to western blotting.
To test the effect of 30-nt binding mode on RPA32 phosphorylation, 75 nM RPA was incubated with 30 nM ssDNA (dT10, dT20, or dT30) in ATR kinase buffer (20 mM HEPES [8.0], 10 mM MgCl2, 2 mM DTT, and 0.1 mM ATP) at 23°C for 20 min. Then, 25 ul of the reactant was added to the immunoprecipitated ATR kinases and incubated at 30°C for 50 min. After the reaction, samples were separated by SDS PAGE and analyzed by western blotting with indicated antibodies.
To test the effect of NSMF on RPA32 phosphorylation, excessive RPA (150 nM) was incubated with 10 nM of 91-nt ssDNA in ATR kinase buffer at 23°C for 20 min, and then 80 nM NSMF was added followed by 30 min incubation. 25 ul of the reactant was added to the immunoprecipitated ATR kinases and incubated at 30°C for 30 min. After the reactions, all samples were separated by SDS PAGE and analyzed by western blotting with indicated antibodies. For each condition, we tested greater than 10 cells and repeated the same experiments three times for one cell. The error bar represents a standard error in triplicate. (C-D) The interaction between NSMF and each RPA subunit using IP in an overexpression system. HEK293T cells were transfected with indicated plasmids for 24 hrs and lysed (C) with or (D) without benzonase. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and analyzed by western blotting with an anti-Myc antibody (αMyc).

II. Supplementary Figures and
(A) Purification of full-length 3xFLAG-NSMF-eGFP. Top: schematic of NSMF construct. Maltose binding protein (MBP) that is eliminated by HRV 3C protease and 3xFLAG is placed at N-terminus, and eGFP is tagged at C-terminus. Bottom: 10% SDS PAGE of purified fulllength NSMF (~90 kDa) and western blotting with antibodies for NSMF (αNSMF), GFP (αGFP), and FLAG (αFLAG). (B) SDS PAGE for purified wild-type (WT) RPA trimer, RPA32 subunit, RPA14 subunit, and RPA-eGFP (eGFP is tagged at RPA70 subunit). (C) In vitro IP for the interaction between NSMF-eGFP and each RPA subunit. RPA70 subunit was purchased (MBS967200, MyBioSource) and RPA32 and RPA14 subunits were purified. Each RPA subunit and NSMF-eGFP were incubated and then immunoprecipitated using anti-GFP antibody. The immunoprecipitants were analyzed by western blotting with indicated antibodies.  Figure 4E. (D) EMSA of NSMF-eGFP with for Cy5-labeled ssDNA to which RPA bound. NSMF-eGFP is stuck in the well without overlapping with ssDNA, indicating that NSMF does not co-bind to the RPA-ssDNA complex.