RNA-binding protein Sam68 effects poly(ADP-ribose) polymerase 1 activity

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Abstract

Taking into account the involvement of RNA-binding proteins in the regulation of the activity of poly(ADP-ribose) polymerase 1 (PARP1), a key factor of DNA repair, the effect of the intrinsically disordered protein Sam68 (Src-associated substrate during mitosis of 68 kDa) on the catalytic activity of this enzyme was studied. Plasmid containing the coding sequence of the Sam68 protein was obtained. Using the obtained construct, the conditions for Sam68 expression in Escherichia coli cells were optimized and a procedure for protein purification was developed. It was found that Sam68 is able to regulate the catalytic activity of PARP1, stimulating auto-poly(ADP-ribosyl)ation of PARP1, interacting with damaged DNA and purified poly(ADP-ribose) (PAR). Based on the experimental data, a hypothesis on the mechanism of the PARP1 activity stimulation by the Sam68 protein was proposed, which consists in the formation of a complex of Sam68 with poly(ADP-ribosyl)ated PARP1. Sam68 interacts with PAR, shielding its negative charge, which increases the time of PARP1 in the complex with damaged DNA and the overall yield of PAR synthetized by this enzyme.

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About the authors

K. N. Naumenko

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences

Email: lavrik@niboch.nsc.ru
Russian Federation, 630090 Novosibirsk

E. A. Berezhnev

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences

Email: lavrik@niboch.nsc.ru
Russian Federation, 630090 Novosibirsk

T. A. Kurgina

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences

Email: lavrik@niboch.nsc.ru
Russian Federation, 630090 Novosibirsk

M. V. Sukhanova

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences

Email: lavrik@niboch.nsc.ru
Russian Federation, 630090 Novosibirsk

O. I. Lavrik

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences

Author for correspondence.
Email: lavrik@niboch.nsc.ru
Russian Federation, 630090 Novosibirsk

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Supplementary files

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2. Fig. 1. Analysis of His-tag-Sam68 expression in the lysate of different producer strains by denaturing gel electrophoresis in 10% SDS-PAGE followed by staining with Coomassie R-250 (a) or Western blot (b). Lanes: 1 and 14 – molecular mass marker (Bio-Rad, USA); 2 and 3 – strain BL21(DE3) before and after induction; 4 and 5 – strain BL21(DE3)GeneX before and after induction; 6 and 7 – strain BL21(DE3)pLysS before and after induction; 8 and 9 – strain Rosetta(DE3) before and after induction; 10 and 11 – strain Rosetta(DE3)pLysS before and after induction. 12 and 13 – Rosetta(DE3)GamiB strain before and after induction

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3. Fig. 2. When expressed in E. coli cells, Sam68 accumulates as inclusion bodies. a – Analysis of the His-tag-Sam68 content in the soluble and insoluble fractions by denaturing gel electrophoresis in 10% SDS-PAGE followed by staining with Coomassie R-250. Lanes: 1 and 8 – molecular weight marker (Bio-Rad); 2 – soluble fraction after centrifugation of lysed cells; 3 – lysate of the producer strain Rosetta(DE3)pLysS; 4 – soluble fraction in 1 M urea after centrifugation of lysed cells; 5 – insoluble fraction; 6 – soluble fraction in 8 M urea; 7 – insoluble fraction after treatment of cell lysate with 8 M urea. b – Analysis of the protein content after chromatography on a column with Ni-NTA. Lanes: 1 – molecular mass marker; 2 – 0.1 μg; 3 – 0.2 μg; 4 – 0.3 μg; 5 – 0.4 μg; 6 – 0.5 μg; 7 – 1 μg of protein obtained after elution with Ni-NTA

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4. Fig. 3. Analysis of Sam68 solubility in urea by denaturing gel electrophoresis in 10% SDS-PAGE followed by staining with Coomassie R-250. Reaction mixtures contained 50 mM Tris-HCl (pH 8.0), 200 mM NaCl, 0.1% NP-40, 7 mM 2-mercaptoethanol, 1 μM Sam68 and urea at the appropriate concentration (125 mM–8 M)

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5. Fig. 4. Analysis of Sam68 solubility at low urea concentration (150 mM) in the presence of arginine (a) or TMAO (b) by denaturing gel electrophoresis in 10% SDS-PAGE followed by Coomassie R-250 staining. The reaction mixture contained 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.1% NP-40, 1 mM DTT, 150 mM urea, 1 μM Sam68 and arginine/TMAO at the indicated concentrations. The soluble fraction (↑) and the precipitate (↓) were analyzed.

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6. Fig. 5. Effect of urea (a and b) and arginine (c and d) on the catalytic activity of PARP1. a – Autoradiograph of 10% SDS-PAGE, in which the separation of the PARylation reaction products was performed in the presence of urea. b – Diagram constructed after analyzing the distribution of radioactivity in the gel shown in panel (a). PARP1 activity in the absence of urea is taken as 100%. c – Autoradiograph of 10% SDS-PAGE, in which the separation of the PARylation reaction products was performed in the presence of arginine. d – Diagram constructed after analyzing the distribution of radioactivity in the gel shown in panel (c). PARP1 activity in the absence of arginine is taken as 100%.

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7. Fig. 6. Sam68 stimulates PARP1 activity. a – Kinetics of PAR synthesis level in the presence of different concentrations of Sam68. The PAR synthesis reaction was carried out using [32P]NAD+ and stopped by transferring aliquots of the reaction mixture to Whatman filter paper soaked in TCA. The graphs show the mean values ​​± standard deviations for three independent experiments. b – Analysis of PARP1 auto-PARylation and Sam68 trans-PARylation by denaturing gel electrophoresis. Autoradiograph of 10% SDS-PAGE, in which the separation of the reaction products of protein PARylation was carried out

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8. Fig. 7. Binding of PAR by Sam68. a – Autoradiograph of native 5% PAGE, in which Sam68•PAR complexes were separated. b – Relative binding of Sam68 to PAR, estimated from the electropherogram analysis shown in panel (a). The affinity (EC50) of Sam68 for PAR was defined as the concentration of Sam68 at which 50% of PAR is in the complex. Reaction mixtures contained 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1 mM DTT, 500 mM urea, 100 μg/ml BSA, 10 nM [32P]-labeled PAR, and Sam68 at the indicated concentrations (75 nM–5 μM)

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9. Fig. 8. Affinity of Sam68 to damaged DNA. Change in DNA fluorescence anisotropy (FAM-Nick) in the presence of different concentrations of Sam68. Reaction mixtures contained 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1 mM DTT, 100 μg/ml BSA, 500 mM urea, 10 nM DNA (FAM-Nick) and Sam68 at the indicated concentrations.

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10. Fig. 9. Study of complex formation of PARP1 and Sam68 with DNA (FAM-Nick) using the fluorescence anisotropy method. Reaction mixtures of 10 μl volume contained 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1 mM DTT, 500 mM urea, 10 nM DNA (FAM-Nick), 0–10 μM Sam68 and 100 nM PARP1

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11. Fig. 10. Hypothetical mechanism of regulation of PARP1 activity by Sam68 protein. a – Formation of a complex of PARP1 with damaged DNA. b – Auto-poly(ADP-ribosyl)ation of PARP1. c – Formation of a complex of Sam68 with PAR covalently attached to PARP1. By binding to PAR covalently attached to PARP1, Sam68 shields the negative charge of this polymer, stabilizing the complex of PARP1 with damaged DNA, and thereby stimulates the synthesis of poly(ADP-ribose).

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12. Appendix
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