The biophysical, biochemical and structural characterization of Poly(ADP-ribose) Polymerase-1 (PARP-1) and its complexes with DNA-damage models and chromatin substrates
Date
2013
Authors
Clark, Nicholas James, author
Luger, Karolin, advisor
Bailey, Susan, committee member
DeLuca, Jennifer, committee member
Hansen, Jeffrey C., committee member
Woody, Robert, committee member
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Abstract
Eukaryotic DNA is highly dynamic and must be compacted and organized with the help of cellular machines, proteins, into 'heterochromatin' state. At its basic level, chromatin is comprised of spool-like structures of protein complexes termed histones, which bind and organize DNA into larger fibrous structures. Cellular processes like transcription and DNA-damage repair require that chromatin be at least partially stripped of its protein components, which in turn allows for complete accessibility by DNA-repair or transcription machinery. A number of protein factors contribute to chromatin structure regulation. Poly(ADP-ribose) Polymerase-1 (PARP-1) is one of these proteins that exists in all eukaryotic organisms except for yeast. In its inactive form, it compacts chromatin, but performs its chromatin-opening function by covalently modifying itself and other nuclear proteins with long polymers of ADP-ribose in response to DNA damage. Thus, it also serves as a first responder to many types of DNA damage. The highly anionic polymers serve to disrupt protein-DNA interactions and thus allow for the creation of a temporary euchromatin structure. Contained herein are investigations aimed at addressing key questions regarding key differences between the interactions of PARP-1 and chromatin and its DNA-damage substrates. Our experiments show that human PARP-1 interacts with and is enzymatically activated to a similar level by a variety of different DNA substrates. In terms of chromatin, it appears that PARP-1 fails to interact with nucleosomes that do not have linker DNA. PARP-1 most effectively interacts with chromatin by simultaneously binding two DNA strands through contacts made by its two N-terminal Zn-finger domains. Small-Angle X-ray (SAXS) and Neutron Scattering (SANS) and molecular dynamics (MD) experiments were combined with biophysical and biochemical studies to better describe the structural effects of DNA binding on PARP-1. The average solution structure of PARP-1 indicates that the enzyme is a monomeric, non-spherical, elongated molecule with a radius of gyration (Rg) of ~55Å. The DNA-bound form of PARP-1 is also monomeric and binding DNA causes the molecule to become more elongated with an average Rg of ~80Å.
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Subject
DNA complexes
SAXS
SANS
nucleosome
PARP-1
chromatin