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Parsing PARP: the enzymatic and biophysical characterization of poly (ADP-Ribose) polymerases I and II

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dc.contributor.authorHepler, Maggie R. D., author
dc.contributor.authorLuger, Karolin, advisor
dc.contributor.authorBailey, Susan, committee member
dc.contributor.authorYao, TingTing, committee member
dc.date.accessioned2015-08-28T14:35:01Z
dc.date.available2016-08-14T06:30:24Z
dc.date.issued2015
dc.description.abstractThe ADP-ribosyl transferase (ART) family is a prominent group of at least seventeen enzymes comprised of mono (ADP-ribose) transferases (MARTs) and poly (ADP-ribose) polymerases (PARPs). Each family member contains a conserved PARP signature motif in the catalytic domain. Enzymatically active proteins, in the presence of co-factor NAD+, catalyze individual or multiple ADP-ribose groups onto themselves or other proteins in automodification and heteromodification, respectively. The act of ADP-ribosylation implicates the ART family in a multitude of cellular processes including, but not limited to, transcription, apoptosis, DNA damage, metabolism, and inflammation. The founding member of the ART family is PARP-1, a first responder to DNA damage and regulator of active gene expression. In its inactive state and as a chromatin architectural protein, PARP-1 tightly binds chromatin, thereby regulating cellular activities, signifying the importance of PARP-1 and chromatin interaction. Importantly, PARP-1 must be activated and automodified in order to bind histones and gain nucleosome assembly function. Structurally similar and in many ways thought to be functionally redundant, PARP-2 is also thought to primarily function in the DNA damage response. PARP-2 has a non-canonical DNA binding domain, and therefore it is able to recognize different types of DNA structures in comparison to PARP-1, which could suggest a unique role for PARP-2 in repair. PARP-2 has not been extensively studied in a chromatin or gene regulation context due to this assumed redundancy. Given the pronounced functional changes in PARP-1 upon automodification, it is important to better understand what exactly triggers its enzymatic activity. Similarly, due to the functional redundancy of PARP-2, insight into activators of its enzymatic activity could indicate specificity and selectivity for the protein. However, determining the details of nuclear components that activate PARP-1 and PARP-2 are limited by the availability of a reliable quantitative and kinetic assay, as well as by the availability of defined substrates. These limitations hinder the separation of potent, and thus biologically relevant, activators from weak or non-specific activators. Utilizing a fluorescence based enzyme assay adapted for this system, kinetic parameters of PARP-1 and PARP-2 allosteric activators are reported here. As proof of principle and to test the reliability of the enzymatic assay, PARP-1 and PARP-2 activity was first tested with nucleic acids and other previously reported activators, such as nucleosomes and histones. Next, potentially novel activators were tested. Notably, PARP-1 is activated in the presence of its enzymatic product, PAR, indicating a mechanism by which PARP-1 could spread at sites of DNA damage and active gene expression. PARP-2 exhibits unique activation and specificity different from that of PARP-1 through its enzymatic preference for RNA. Further, PARP-1 remains the prominent chromatin related PARP due to the weak interaction, both activity and affinity, of chromatin with PARP-2. However, while PARP-1 and PARP-2 can act individually, affinity and activity studies demonstrate a PARP-1 and PARP-2 complex suggesting that these proteins can act sequentially and simultaneously with one another during a PAR-mediated recruitment and signaling cascade. Overall, these data indicate novel functions and mechanisms for PARP-1 and PARP-2 within the nucleus as critical responders to DNA damage and gene regulation.
dc.format.mediumborn digital
dc.format.mediummasters theses
dc.identifier.urihttp://hdl.handle.net/10217/167077
dc.languageEnglish
dc.language.isoeng
dc.publisherColorado State University. Libraries
dc.relation.ispartof2000-2019
dc.rightsCopyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright.
dc.subjectFRET
dc.subjectPARP-2
dc.subjectPARP-1
dc.subjectenzymology
dc.titleParsing PARP: the enzymatic and biophysical characterization of poly (ADP-Ribose) polymerases I and II
dc.typeText
dcterms.embargo.expires2016-08-14
dcterms.embargo.terms2016-08-14
dcterms.rights.dplaThis Item is protected by copyright and/or related rights (https://rightsstatements.org/vocab/InC/1.0/). You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s).
thesis.degree.disciplineBiochemistry and Molecular Biology
thesis.degree.grantorColorado State University
thesis.degree.levelMasters
thesis.degree.nameMaster of Science (M.S.)

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