Thesis
In vitro binding of base excision repair glycosylases to poly(adp-ribose)
Washington State University
Master of Science (MS), Washington State University
2008
Handle:
https://hdl.handle.net/2376/105430
Abstract
After genomic DNA is damaged, one of the earliest eukaryotic cellular events is the initiation of Poly(ADP-ribose)polymerase-1 (PARP-1) enzymatic activity. PARP-1 converts NAD+ to poly(ADP-ribose) polymers (PAR) which covalently modify several nuclear proteins, most notably histone proteins and PARP-1 itself. PARP-1 and its enzymatic activity are known to be critical for the proper functioning of Base Excision Repair (BER), the primary cellular pathway involved in the repair of DNA single strand breaks and damaged nucleobase products. However, the precise roles that PARsylation plays in BER are not currently clear. In addition to being a posttranslational modification of nuclear proteins, PAR also serves as a binding scaffolding for many proteins via non-covalent, highly specific molecular contacts between protein motifs and PAR. Since the BER proteins XRCC1 and DNA Ligase III have previously been shown to associate with PAR, it is thought that PAR-protein interactions play a role in latter stages of BER. In this thesis, the hypothesis was tested that BER glycosylases, the enzymes involved in the earliest stages of BER, would bind to PAR as well. After the identification of putative PAR-binding sequences in the primary structure of five DNA glycosylases (OGG1, NEIL1, NTH1, MYH, and MPG) slot blot and phenol partitioning assays were carried out on the glycosylase peptides. These assays showed that peptides from OGG1, NTH1, NEIL1, and MPG bind to PAR. The native glycosylase proteins OGG1 and NEIL1 were also observed to bind to PAR, indicating that these glycosylases may interact with PAR in vivo. These PAR-glycosylase interactions have allowed us to propose a new model for the regulation of the earliest stages of BER by PAR. Finally, we have developed a new assay based on formaldehyde crosslinking to assess protein-PAR interactions. This technique is envisioned to facilitate future in vivo studies of PAR-protein interactions in the human cell nucleus following DNA damage.
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Details
- Title
- In vitro binding of base excision repair glycosylases to poly(adp-ribose)
- Creators
- Joseph A. Nichols
- Contributors
- William B. Davis (Degree Supervisor)
- Awarding Institution
- Washington State University
- Academic Unit
- Molecular Biosciences, School of
- Theses and Dissertations
- Master of Science (MS), Washington State University
- Publisher
- Washington State University; [Pullman, Washington] :
- Identifiers
- 99900525008201842
- Language
- English
- Resource Type
- Thesis