Damage to DNA from a variety of sources can lead to damaged proteins, genomic instability, aneuploidy, and cancer. It is therefore essential to repair DNA damage, and to do so a variety of DNA repair mechanisms have evolved. One of the repair mechanisms, known as homologous recombination (HR) repair, uses an undamaged sister chromatid as a template to make error free repairs to double-strand (ds) DNA breaks. While many proteins are involved in HR, this work focuses on testing the interactions of a subset of these proteins known as the Rad51 paralogs.
The goal of this study is to determine if the putative Rad51 paralogs in Drosophila melanogaster are sufficiently conserved as to function in the same manner as their human counterparts. This research is part of a larger project to determine if Drosophila melanogaster is a good model organism for studying HR in humans (Hs).
The D. melanogaster Rad51 gene, and its four paralogs Spn D, Spn B, Rad51D, XRCC2 (the last 2 identified by sequence homology), and human hsRad51D and hsXRCC2, were cloned into Invitrogen's TOPO protein expression vector. When induced with IPTG, the resulting fusion proteins contains either aN-terminal Xpress TM epitope or a C-terminal V5 epitope. The fusion proteins were used in immunoprecipitation assays with antibodies against the epitope tags to test for proteinprotein interactions.
While many of the assays were inconclusive and are still being optimized, the interaction of the C-terminally tagged dmXRCC2 with theN-terminally tagged hsRad51D gave a positive result. This single interspecies result suggests that homologous recombination is highly conserved between D. melanogaster and humans.
| Identifer | oai:union.ndltd.org:pacific.edu/oai:scholarlycommons.pacific.edu:uop_etds-1750 |
| Date | 01 January 2010 |
| Creators | Buffleben, George M. |
| Publisher | Scholarly Commons |
| Source Sets | University of the Pacific |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | University of the Pacific Theses and Dissertations |
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