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

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.

Authors:
; ; ; ; ; ;  [1];  [2];  [2];  [2]
  1. Children
  2. (
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
National Institutes of Health (NIH)
OSTI Identifier:
1418030
Resource Type:
Journal Article
Resource Relation:
Journal Name: Methods Mol. Biol.; Journal Volume: 1608; Journal Issue: 2017
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Chassé, Maggie H., Muthurajan, Uma M., Clark, Nicholas J., Kramer, Michael A., Chakravarthy, Srinivas, Irving, Thomas, Luger, Karolin, IIT), Colorado), and Amgen). Biochemical and Biophysical Methods for Analysis of Poly(ADP-Ribose) Polymerase 1 and Its Interactions with Chromatin. United States: N. p., 2018. Web. doi:10.1007/978-1-4939-6993-7_16.
Chassé, Maggie H., Muthurajan, Uma M., Clark, Nicholas J., Kramer, Michael A., Chakravarthy, Srinivas, Irving, Thomas, Luger, Karolin, IIT), Colorado), & Amgen). Biochemical and Biophysical Methods for Analysis of Poly(ADP-Ribose) Polymerase 1 and Its Interactions with Chromatin. United States. doi:10.1007/978-1-4939-6993-7_16.
Chassé, Maggie H., Muthurajan, Uma M., Clark, Nicholas J., Kramer, Michael A., Chakravarthy, Srinivas, Irving, Thomas, Luger, Karolin, IIT), Colorado), and Amgen). Thu . "Biochemical and Biophysical Methods for Analysis of Poly(ADP-Ribose) Polymerase 1 and Its Interactions with Chromatin". United States. doi:10.1007/978-1-4939-6993-7_16.
@article{osti_1418030,
title = {Biochemical and Biophysical Methods for Analysis of Poly(ADP-Ribose) Polymerase 1 and Its Interactions with Chromatin},
author = {Chassé, Maggie H. and Muthurajan, Uma M. and Clark, Nicholas J. and Kramer, Michael A. and Chakravarthy, Srinivas and Irving, Thomas and Luger, Karolin and IIT) and Colorado) and Amgen)},
abstractNote = {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.},
doi = {10.1007/978-1-4939-6993-7_16},
journal = {Methods Mol. Biol.},
number = 2017,
volume = 1608,
place = {United States},
year = {Thu Jan 18 00:00:00 EST 2018},
month = {Thu Jan 18 00:00:00 EST 2018}
}
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  • Poly(ADP-ribose) polymerase-1 (PARP-1) has two homologous zinc finger domains, Zn1 and Zn2, that bind to a variety of DNA structures to stimulate poly(ADP-ribose) synthesis activity and to mediate PARP-1 interaction with chromatin. The structural basis for interaction with DNA is unknown, which limits our understanding of PARP-1 regulation and involvement in DNA repair and transcription. Here, we have determined crystal structures for the individual Zn1 and Zn2 domains in complex with a DNA double strand break, providing the first views of PARP-1 zinc fingers bound to DNA. The Zn1-DNA and Zn2-DNA structures establish a novel, bipartite mode of sequence-independent DNAmore » interaction that engages a continuous region of the phosphodiester backbone and the hydrophobic faces of exposed nucleotide bases. Biochemical and cell biological analysis indicate that the Zn1 and Zn2 domains perform distinct functions. The Zn2 domain exhibits high binding affinity to DNA compared with the Zn1 domain. However, the Zn1 domain is essential for DNA-dependent PARP-1 activity in vitro and in vivo, whereas the Zn2 domain is not strictly required. Structural differences between the Zn1-DNA and Zn2-DNA complexes, combined with mutational and structural analysis, indicate that a specialized region of the Zn1 domain is re-configured through the hydrophobic interaction with exposed nucleotide bases to initiate PARP-1 activation.« less
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