Functional Roles of PARP2 in Assembling Protein–Protein Complexes Involved in Base Excision DNA Repair
Abstract
:1. Introduction
2. Results
2.1. Detection and Quantification of PARP2–Protein Interactions by Fluorescence Titration and FRET Experiments
2.2. DLS Study of Oligomerization States of PARP2 and Its Hetero-Associated Complexes with BER Proteins
2.3. DLS Detection of Association of Poly(ADP-ribosyl)ated PARP2 in Solution
2.4. Visualization of PARylated Protein Associates Formed by PARP1 and PARP2
3. Discussion
4. Materials and Methods
4.1. Protein Expression and Purification
4.2. Oligonucleotide Substrates
4.3. Dynamic Light-Scattering (DLS) Studies of PARP2 and Its Protein Complexes, and PARP2-Catalysed Poly(ADP-ribosyl)ation
4.4. Fluorescent Labelling of APE1, Polβ, PARP1, XRCC1, and PARP2
4.5. Fluorescence Studies of PARP2 Interaction with Proteins and DNAs
4.6. Visualization of PARP1 and PARP2 Associates Formed upon PARylation Reaction
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
PARP1 | poly(ADP)ribose polymerase 1 |
PARP2 | poly(ADP)ribose polymerase 2 |
Polβ | DNA polymerase β |
XRCC1 | X-ray repair cross-complementing protein 1 |
APE1 | apurinic/apyrimidinic endonuclease 1 |
BER | base excision repair |
SSB | single-strand break |
FAM | 5(6)-carboxyfluorescein |
AF488 | 5-Alexa Fluor 488 |
Cy3 | sulfo-Cyanine 3 |
Cy5 | sulfo-Cyanine 5 |
SE | N-succinimidyl ester |
FRET | fluorescence resonance energy transfer |
DLS | dynamic light scattering |
PAR | poly(ADP-ribose) |
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Labelled Protein 1 | Protein Partner | EC50 2, nM | E 3 |
---|---|---|---|
FAM-PARP2 | PARP2 | 152 ± 12 | |
FAM-PARP2 | Polβ | 185 ± 18 * | |
FAM-PARP2 | XRCC1 | 190 ± 18 * | |
FAM-PARP2 | PARP1 | 146 ± 10 | |
FAM-APE1 | PARP2 | 61 ± 8 *** | |
AF-PARP2 | PARP1 | 79 ± 7 *** | |
AF-PARP2 | Cy3-PARP1 | 76 ± 7 *** | 0.36 ± 0.04 |
Cy3-PARP1 | PARP2 | 90 ± 6 *** | |
Cy3-XRCC1 | PARP2 | 196 ± 10 ** |
Labelled Protein 1 | DNA 2 | Protein Partner | EC50 3, nM | Effect on Binding Affinity 4 | Effect on FRET Efficiency 5 |
---|---|---|---|---|---|
FAM-PARP2 | gap-DNA | PARP2 | 101 ± 9 ** | 1.5 (+) | |
FAM-PARP2 | gap-DNA | Polβ | 106 ± 10 ** | 1.7 (+) | |
FAM-PARP2 | gap-DNA | XRCC1 | 164 ± 12 * | 1.2 (+) | |
AF-PARP2 | nick-DNA | PARP1 | 44 ± 5 ** | 1.8 (+) | |
AF-PARP2 | ds-DNA | PARP1 | 144 ± 16 ** | 1.8 (−) | |
AF-PARP2 | nick-DNA | Cy3-PARP1 | −0.04 * | ||
AF-PARP2 | ds-DNA | Cy3-PARP1 | +0.08 ** | ||
FAM-PARP2 | gap-DNA | ≤18 | |||
AF-PARP2 | nick-DNA | ≤15 | |||
AF-PARP2 | ds-DNA | 35 ± 4 |
Protein(s) 1 (Mw 2, kDa) | DLS RH 3, nm | Theoretical RH 4, nm | Oligomeric State 5 | |
---|---|---|---|---|
Protomer | Dimer (Tetramer) | |||
Homo- and hetero-oligomerization of PARP2 | ||||
PARP2 (62.0) | 5.0 ± 0.4 | 3.47 | 4.67 (6.28) | A2 |
PARP2 + Polβ (100.3) | 5.6 ± 0.3 | 4.27 | 5.74 | (AB)2 |
PARP2 + APE1 (97.5) | 4.9 ± 0.2 | 4.21 | 5.67 | AB+(AB)2 |
PARP2 + XRCC1 (131.5) | 7.6 ± 0.3 | 4.79 | 6.44 (8.66) | (AB)2 |
PARP2 + PARP1 (175.0) | 7.8 ± 0.6 | 5.41 | 7.28 | (AB)2 |
Homo-oligomerization of BER proteins determined in our previous study [24] | ||||
Polβ (38.3) | 3.5 ± 0.2 | 2.83 | 3.80 | A2 |
APE1 (35.5) | 3.2 ± 0.2 | 2.74 | 3.68 | A2 |
XRCC1 (69.5) | 5.7 ± 0.2 | 3.65 | 4.90 | A2 |
PARP1 (113.0) | 8.0 ± 0.5 | 4.50 | 6.04(8.12) | A2 6 |
Protein(s) 1 | RH 2, (nm) | |||
---|---|---|---|---|
Before Reaction Initiation | PARylated Protein Associate 3 | PARylated Protein after Addition of EDTA 3 | PARylated Protein after PARG Treatment 3 | |
PARP2 | 5.0 ± 0.4 | 383 ± 45 | 5.6 ± 0.3 | 5.2 ± 0.3 |
PARP2 + Polβ | 5.6 ± 0.3 | 370 ± 43 | 6.2 ± 0.5 | 5.8 ± 0.2 |
PARP2 + APE1 | 4.9 ± 0.2 | 440 ± 51 | 5.2 ± 0.3 | n. d. 4 |
PARP2 + XRCC1 | 7.6 ± 0.3 | 11.3 ± 1.6 | 6.7 ± 0.5 | 5.8 ± 0.4 |
PARP2 + PARP1 | 7.8 ± 0.4 | 14.8 ± 1.4 | 9.9 ± 1.2 | n. d. 4 |
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Vasil’eva, I.; Moor, N.; Anarbaev, R.; Kutuzov, M.; Lavrik, O. Functional Roles of PARP2 in Assembling Protein–Protein Complexes Involved in Base Excision DNA Repair. Int. J. Mol. Sci. 2021, 22, 4679. https://doi.org/10.3390/ijms22094679
Vasil’eva I, Moor N, Anarbaev R, Kutuzov M, Lavrik O. Functional Roles of PARP2 in Assembling Protein–Protein Complexes Involved in Base Excision DNA Repair. International Journal of Molecular Sciences. 2021; 22(9):4679. https://doi.org/10.3390/ijms22094679
Chicago/Turabian StyleVasil’eva, Inna, Nina Moor, Rashid Anarbaev, Mikhail Kutuzov, and Olga Lavrik. 2021. "Functional Roles of PARP2 in Assembling Protein–Protein Complexes Involved in Base Excision DNA Repair" International Journal of Molecular Sciences 22, no. 9: 4679. https://doi.org/10.3390/ijms22094679