Non-Classical H1-like PARP1 Binding to Chromatosome
Abstract
1. Introduction
2. Materials and Methods
2.1. DNA Templates
2.2. Protein Purification
2.3. Assembly of Nucleosomes
2.4. Sample Preparations
2.5. SpFRET Experiments
2.6. Electrophoretic Mobility Shift Assay (EMSA)
2.7. Native Gel Electrophoresis of Proteins in Agarose
2.8. Western Blotting
2.9. Molecular Modeling
3. Results
3.1. PARP1 Forms Three Types of Complexes with 2LN Nucleosomes and Reorganizes Nucleosome Structure
3.2. Poly-ADP-Ribosylation Causes Differential Dissociation of PARP1 Molecules from 2LN-PARP1 Complexes
3.3. Can PARP1 Reorganize the Structure of a Chromatosome?
3.4. Molecular Modeling of the PARP1 Binding to the Core Region of a Nucleosome near the Nucleosome Dyad
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
BRCT | domain of PARP1 containing BRCA1 C terminus motif |
CAT | catalytic domain of PARP1 |
Cy3 and Cy5 | fluorescent dyes cyanine 3 and cyanine 5 |
DSBs | double-strand breaks |
EMSA | electrophoretic mobility shift assay |
EPR- | proximity ratio (FRET efficiency without correction for detection sensitivity and quantum yields of fluorophores). |
FRET | Forster resonance energy transfer |
PARP1 | poly(ADP-ribose)polymerase 1 |
NAD+ | β-nicotinamide adenine dinucleotide |
NPS | nucleosome positioning sequence |
spFRET | single particle Forster resonance energy transfer |
2LNP | nucleosomes that were assembled on the 187 bp DNA template containing the 147 bp Widom 603-42A NPS flanked by a pair of 20 bp linkers. Cy3 and Cy5 labels were attached to thymine bases at positions 13 and 91 bp from the beginning of NPS |
2LNM | nucleosomes that were assembled on the 187 bp DNA template containing the 147 bp Widom 603-42A NPS flanked by a pair of 20 bp linkers. Cy3 and Cy5 labels were attached to thymine bases at positions 35 and 112 bp from the beginning of NPS |
2LND | nucleosomes that were assembled on the 187 bp DNA template containing the 147 bp Widom 603-42A NPS flanked by a pair of 20 bp linkers. Cy3 and Cy5 labels were attached to thymine bases at positions 57 and 135 bp from the beginning of NPS. |
2LNL | nucleosomes that were assembled on the 187 bp DNA template containing the 147 bp Widom 603-42A NPS flanked by a pair of 20 bp linkers. Cy3 and Cy5 labels were positioned in linker DNA fragments at a distance of 18 bp before and after NPS |
2LNL2 | nucleosomes that were assembled on the 227 bp DNA template containing the 147 bp Widom 603-42A NPS flanked by a pair of 40 bp linkers. Cy3 and Cy5 labels were positioned in linker DNA fragments at a distance of 25 bp before and after NPS |
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Koshkina, D.; Maluchenko, N.; Nilov, D.; Lyubitelev, A.; Korovina, A.; Pushkarev, S.; Armeev, G.; Kirpichnikov, M.; Studitsky, V.; Feofanov, A. Non-Classical H1-like PARP1 Binding to Chromatosome. Cells 2025, 14, 1309. https://doi.org/10.3390/cells14171309
Koshkina D, Maluchenko N, Nilov D, Lyubitelev A, Korovina A, Pushkarev S, Armeev G, Kirpichnikov M, Studitsky V, Feofanov A. Non-Classical H1-like PARP1 Binding to Chromatosome. Cells. 2025; 14(17):1309. https://doi.org/10.3390/cells14171309
Chicago/Turabian StyleKoshkina, Darya, Natalya Maluchenko, Dmitry Nilov, Alexander Lyubitelev, Anna Korovina, Sergey Pushkarev, Grigoriy Armeev, Mikhail Kirpichnikov, Vasily Studitsky, and Alexey Feofanov. 2025. "Non-Classical H1-like PARP1 Binding to Chromatosome" Cells 14, no. 17: 1309. https://doi.org/10.3390/cells14171309
APA StyleKoshkina, D., Maluchenko, N., Nilov, D., Lyubitelev, A., Korovina, A., Pushkarev, S., Armeev, G., Kirpichnikov, M., Studitsky, V., & Feofanov, A. (2025). Non-Classical H1-like PARP1 Binding to Chromatosome. Cells, 14(17), 1309. https://doi.org/10.3390/cells14171309