PMCID: PMC5695667 DOI: 10.1007/978-1-4939-6993-7_16
Poly (ADP-Ribose) Polymerase I (PARP-1) is a first responder to DNA damage and participates in the regulation of gene expression. The interaction of PARP-1 with chromatin and DNA is complex and involves at least two different modes of interaction. In its enzymatically inactive state, PARP-1 binds native chromatin with similar affinity as it binds free DNA ends. Automodification of PARP-1 affects interaction with chromatin and DNA to different extents. Here we describe a series of biochemical and biophysical techniques to quantify and dissect the different binding modes of PARP-1 with its various substrates. The techniques listed here allow for high throughput and quantitative measurements of the interaction of different PARP-1 constructs (inactive and automodified) with chromatin and DNA damage models.
Methods in molecular biology (Clifton, N.J.)
Animals; Chromatin; DNA; DNA Damage; Electrophoretic Mobility Shift Assay; Humans; Microscopy, Atomic Force; Poly(ADP-ribose) Polymerases; Protein Binding; Ultracentrifugation
Analytical ultracentrifugation; Atomic force microscopy; Electrophoretic mobility shift assays; HI-FI FRET; Job plot; Multi-angle light scattering; PARP-1; Small angle X-ray scattering
Chassé, Maggie H.; Muthurajan, Uma M.; Clark, Nicholas J.; Kramer, Michael A.; Chakravarthy, Srinivas; Irving, Thomas; and Luger, Karolin, "Biochemical and Biophysical Methods for Analysis of Poly(ADP-Ribose) Polymerase 1 and Its Interactions with Chromatin." (2017). Manuscripts, Articles, Book Chapters and Other Papers. 1157.