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Methods Mol Biol. 2017;1608:231-253. doi: 10.1007/978-1-4939-6993-7_16.

Biochemical and Biophysical Methods for Analysis of Poly(ADP-Ribose) Polymerase 1 and Its Interactions with Chromatin.

Author information

1
Department of Chemistry and Biochemistry, University of Colorado at Boulder, 3415 Colorado Avenue, Boulder, CO, 80303, USA.
2
Van Andel Research Institute, Grand Rapids, MI, USA.
3
Amgen Inc., Thousand Oaks, CA, USA.
4
Children's Mercy Hospital, Kansas City, MO, USA.
5
BioCAT, CSRRI and Department BCS, Illinois Institute of Technology, Chicago, IL, USA.
6
Department of Chemistry and Biochemistry, University of Colorado at Boulder, 3415 Colorado Avenue, Boulder, CO, 80303, USA. karolin.luger@colorado.edu.
7
Howard Hughes Medical Institute, Chevy Chase, MD, USA. karolin.luger@colorado.edu.
8
Institute for Genome Architecture and Function, Colorado State University, Fort Collins, CO, USA. karolin.luger@colorado.edu.

Abstract

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.

KEYWORDS:

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

PMID:
28695514
PMCID:
PMC5695667
DOI:
10.1007/978-1-4939-6993-7_16
[Indexed for MEDLINE]
Free PMC Article

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