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, 292 (10), 4313-4325

Ribosomal Protein S3 Negatively Regulates Unwinding Activity of RecQ-like Helicase 4 Through Their Physical Interaction

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Ribosomal Protein S3 Negatively Regulates Unwinding Activity of RecQ-like Helicase 4 Through Their Physical Interaction

Ajay Vitthal Patil et al. J Biol Chem.

Abstract

Human RecQ-like helicase 4 (RECQL4) plays crucial roles in replication initiation and DNA repair; however, the contextual regulation of its unwinding activity is not fully described. Mutations in RECQL4 have been linked to three diseases including Rothmund-Thomson syndrome, which is characterized by osteoskeletal deformities, photosensitivity, and increased osteosarcoma susceptibility. Understanding regulation of RECQL4 helicase activity by interaction partners will allow deciphering its role as an enzyme and a signaling cofactor in different cellular contexts. We became interested in studying the interaction of RECQL4 with ribosomal protein S3 (RPS3) because previous studies have shown that RPS3 activity is sometimes associated with phenotypes mimicking those of mutated RECQL4. RPS3 is a small ribosomal protein that also has extraribosomal functions, including apurnic-apyrimidinic endonuclease-like activity suggested to be important during DNA repair. Here, we report a functional and physical interaction between RPS3 and RECQL4 and show that this interaction may be enhanced during cellular stress. We show that RPS3 inhibits ATPase, DNA binding, and helicase activities of RECQL4 through their direct interaction. Further domain analysis shows that N-terminal 1-320 amino acids of RECQL4 directly interact with the C-terminal 94-244 amino acids of RPS3 (C-RPS3). Biochemical analysis of C-RPS3 revealed that it comprises a standalone apurnic-apyrimidinic endonuclease-like domain. We used U2OS cells to show that oxidative stress and UV exposure could enhance the interaction between nuclear RPS3 and RECQL4. Regulation of RECQL4 biochemical activities by RPS3 along with nuclear interaction during UV and oxidative stress may serve to modulate active DNA repair.

Keywords: DNA helicase; DNA repair; base excision repair (BER); genomic instability; oxidative stress; protein-protein interaction.

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

FIGURE 1.
FIGURE 1.
Purification and biochemical analysis of human RECQL4 helicase and K508N-RECQL4 mutant. A, schematic of the recombinant human RECQL4 construct expressed in SF9 cells with a C-terminal His tag and an N-terminal GST tag attached to RECQL4 by the PreScission protease cleavage sequence. * represents position of the K508N mutation for K508N-RECQL4 purification. B, purification strategy for RECQL4 and K508N-RECQL4. C, purification stage fractions of RECQL4 protein. Total lysate, total lysate of protein expressing SF9 cells; Clarified lysate, total lysate supernatant after centrifugation; Post PPT, clarified lysate applied to glutathione column followed by PreScission protease treatment (PPT); Post Nickel, nickel column concentration of the protein in the supernatant of the post PPT fraction. D, final post nickel purification fraction of K508N-RECQL4 (purification columns and process, the same as RECQL4). E, ATPase assay. The plot depicts percentage of [γ-32P]ATP hydrolyzed in the presence of 40 nm RECQL4 and DNA, as well as with no-enzyme and no-DNA controls at indicated time points assayed by TLC. The data points are the mean values of triplicate experiments with S.D. shown as error bars. F, bar graph showing percentages of [γ-32P]ATP hydrolyzed in the presence of 40 nm RECQL4 or the helicase-dead mutant K508N. G, polyacrylamide gel showing DNA helicase activity of RECQL4 and K508N on 5′-FAM-labeled 3′ overhang substrate (20 nm). Substrate and product structures are illustrated on the right side of the gel. * represents the position of FAM label on the substrate.
FIGURE 2.
FIGURE 2.
RECQL4 physically interacts with RPS3. A, nuclear lysates of 293 cell lines stably expressing RECQL4-V5-his or control plasmid are used for V5 pulldown followed by anti-RPS3, anti-V5 and anti-Lamin B immunoblotting. B, Coomassie Blue-stained SDS-PAGE depicting final purified protein fraction of GST-FL-RPS3 after BiorexTM70 and glutathione-Sepharose purification steps. C, GST pulldown assay. Anti-RECQL4 and anti-GST blots show input and pulldown fractions of GST pulldown assay carried out by incubating RECQL4-his with GST-RPS3 or GST only proteins. D, confocal images of representative U2OS cells immunostained by anti-RECQL4, anti-RPS3 antibodies, and DAPI. The plot shows nuclear RPS3 (%) and nuclear colocalization of RPS3-RECQL4 (%) quantified from three-dimensional reconstructions of Z section confocal images, using Imaris software. The data are shown as the means ± S.D. of triplicate experiments.
FIGURE 3.
FIGURE 3.
RPS3 inhibits helicase and ATPase activity of RECQL4. A, representative PAGE showing the RECQL4 helicase assay performed in the presence or absence of indicated concentrations of FL-RPS3 (1–244 aa). All helicase reactions contain 50 nm RECQL4 protein and 20 nm 5′FAM-labeled 3′ overhang substrate. Substrate and product structures are illustrated on the left side of the gel. * represents position of the FAM label on the substrate. Bar graph depicting percentage unwinding against the concentration of RPS3 protein in the helicase reaction. The data points shown are the means of triplicate experiments with S.D. indicated as error bars. B, ATPase activity of RECQL4 assayed using TLC. Of a total 20-μl reaction, 1 μl (with 400 nm RPS3, 80 nm of RECQL4, and 3.4 nm [γ-32P]ATP substrate) was taken for TLC analysis at indicated time points. Bar graph showing percentage of [γ-32P]ATP hydrolyzed in the presence (dotted bars) or absence (shaded bars) of FL-RPS3 plotted against the reaction time course. The data points shown are the means of triplicate experiments with S.D. indicated as error bars.
FIGURE 4.
FIGURE 4.
The C-terminal (94–244 aa) of RPS3 and N-terminal (1–320 aa) of RECQL4 are the main interaction domains of the two proteins. A, schematic showing GST-tagged proteins used for the pulldown assay: GST-FL-RPS3 (1–244 aa), GST-N-RPS3-his (1–94 aa), GST-C-RPS3-his (94–244 aa), and GST only. B, GST pulldown assay. Anti-RECQL4 and anti-GST immunoblots show input and pulldown fractions when FL-RECQL4-V5-his was incubated with GST tagged FL-RPS3, N-RPS3, C-RPS3, or GST only proteins. C, schematic showing V5 tagged proteins used for pulldown assay: FL-RECQL4-V5-his (1–1208 aa), N-RECQL4-V5-his (1–320 aa), and C-RECQL4-V5-his (314–1208 aa). D, V5 pulldown assay. Anti-V5 and anti-RPS3 immunoblots show input and pulldown fractions when GST-FL-RPS3 was incubated with FL-RECQL4-V5-his, N-RECQL4-V5-his, or C-RECQL4-V5-his proteins.
FIGURE 5.
FIGURE 5.
C-RPS3 interacts with N-RECQL4 in vivo. A, 293 cells stably expressing FL-RECQL4-V5-his or N-RECQL4-V5-his transfected with HA-FL-RPS3 (1–244 aa), HA-N-RPS3 (1–94 aa), and HA-C-RPS3 (94–244 aa) plasmid constructs. Whole cell lysates were subjected to V5-IP followed by anti-HA, anti-V5, and anti-GAPDH Western blotting analysis of lysate and IP fractions. B, schematic showing results of V5 coimmunoprecipitation experiments.
FIGURE 6.
FIGURE 6.
C-RPS3 (94–244 aa), but not N-RPS3 (1–94 aa), contributes to RECQL4 helicase activity inhibition. All helicase reactions contain 50 nm RECQL4 protein and 20 nm 5′FAM-labeled 3′ overhang substrate. Substrate and product structures are illustrated on the right side of the gels. * represents position of FAM label on the substrate. A, representative PAGE showing the RECQL4 helicase assay performed in the presence or absence of indicated concentrations of full-length FL-RPS3 (1–244 aa). B, representative PAGE showing the RECQL4 helicase assay performed in the presence or absence of indicated concentrations of C-RPS3 (94–244 aa). C, representative PAGE showing RECQL4 helicase assay performed in the presence or absence of indicated concentrations of N-RPS3 (1–94 aa). D, graph depicting percentage unwinding by RECQL4 plotted against final concentration of FL-RPS3, N-RPS3, or C-RPS3 in RECQL4 helicase reaction. The data points shown are the means of triplicate experiments with S.D. indicated as error bars.
FIGURE 7.
FIGURE 7.
FL-RPS3 and C-RPS3 exhibit comparable AP endonuclease activities. A, representative gel showing plasmid nicking assay. 4 nm depurinated pHC624 substrate was incubated with indicated concentrations of FL-RPS3 (lanes 3–6), N-RPS3 (lanes 7–10), C-RPS3 (lanes 11–14), and APE1 (lanes 1 and 16). Non-depurinated plasmid substrates (4 nm) were used as negative controls (lanes 15–19). Nicked indicates product amount, and Supercoiled indicates substrate amount in the reaction. B, plot showing percentage product formed against concentration of RPS3 proteins in the reaction (representative experiment in A). The data points shown are the means of triplicate experiments with S.D. indicated as error bars. C, gel showing plasmid nicking assay. The gel reveals the effect of indicated concentrations of RECQL4 (lanes 5–8) on AP endonuclease activity of RPS3 along with control reactions. APE1 acted as a positive control (lane 2). 1 unit (1U) is equal to 2 nm final concentration.
FIGURE 8.
FIGURE 8.
Nuclear RECQL4-RPS3 interaction is enhanced under hydrogen peroxide- and UV-induced genotoxic stress in U2OS cells. U2OS cells subjected to mock, hydrogen peroxide (200 or 500 μm), or UV (20 or 40 J/m2) treatment were immunostained with anti-RECQL4 and anti-RPS3 antibodies. Representative confocal images of cells after treatments are shown in the top panels. Plots at the bottom show quantification of nuclear RPS3 (%) and nuclear colocalization of RECQL4-RPS3 (%) carried out using Imaris 8.3.1 software on z sections of confocal images taken from immunostained U2OS cells. The data points shown are the means of triplicate experiments with S.D. indicated as error bars.

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