DNA mismatch repair proteins in Tetrahymena thermophila
Date
2010-04-08T18:58:36Z
Authors
Kudynska, Kate
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Abstract
DNA mismatch repair (MMR) is an essential part of genomic stability, guarding the integrity of the genome in virtually all cells. MMR corrects mismatched bases in DNA and is one of several DNA repair pathways conserved from bacteria to humans. In Escherichia coli, MutS and MutL are the key proteins in MMR. In prokaryotes, the MMR proteins function as homodimers whereas in eukaryotes the MMR proteins come together as heterodimers. In the absence of MMR there is a great increase in mutation frequency and a higher susceptibility to cancer in mammals. Previous studies in our laboratory have identified five MutS homologs and two MutL homologs in the single-celled ciliated protozoan, Tetrahymena thermophila. MMR repair has been extensively studied in E. coli but less is known in eukaryotes.
T. thermophila 's biology and the recent sequencing of its genome make it an attractive eukaryotic research model. In this study, poly-histidine tagged MMR genes from T. thermophila and human thymine DNA glycosylase (TDG) were cloned into two different types of T thermophila expression plasmid. The integrated homologously recombinational T. thermophila vector approach and the espisomal rDNA vector approach which utilized Gateway® technology
a cloning method based on the bacteriophage lambda site-specific recombination system. The homologous vector approach relies on mutant strains of T. thermophila harboring a negatively selectable allele of a P-tubulin gene producing sensitivity to paclitaxol. Upon knocking out the mutant J3-tubulin with the gene of interest the resistance of the Tetrahymena strain is then resorted and selected for. The various T. thermophila expression plasmids and DNA transformations techniques to follow such as biolistic bombardment and conjugated electroporation were carried out in order to optimize the technical aspect of working with T. thermophila. and finally lead to the purification of histidine tagged T. thermophila MMR proteins for antibody production and further studies. This study will lead to insight into the inner workings of DNA mismatch repair and technical aspect of working with the organism and will permit functional and structural studies of the MMR homolog proteins in T. thermophila.
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Keywords
DNA repair, Tetrahymena thermophila, Protozoa