Investigating Replication Protein A's Role in Telomere Maintenance in Arabidopsis thaliana

Abstract

Replication protein A (RPA) and CST are two highly similar heterotrimeric telomere complexes involved in various aspects of DNA metabolism. RPA functions in genome-wide DNA metabolism (including DNA replication, repair and recombination) while CST has been shown to play a specific role in telomere protection and maintenance. Recent studies have implicated RPA in telomere maintenance. Two CST subunits, STN1 and TEN1, have been found to form a sub-complex independent of the CST complex. Given the high level of structural conservation between RPA and CST, I hypothesize that individual CST and RPA subunits form alternative complexes. The aim of this study is to test the hypothesis by determining if RPA and CST subunits associate with each other using an in vitro co-immunoprecipitation assay. Expression vectors have been constructed with RPA2A, RPA2B, RPA3A, RPA3B, STN1, and TEN1 and have been tested in rabbit reticulocyte lysate for protein expression. If association between CST and RPA subunits is found, further studies could investigate how and why the CST and RPA complexes work together to maintain telomeres and to promote aspects of genome stability. Another goal of this study is to construct fluorescent protein tagged RPA1B (GFP) and RPA1D (YFP) for use in subcellular localization studies in Arabidopsis thaliana.