Honors College Thesis


Repression of DNA Mismatch Repair (MMR) in Arabidopsis thaliana through Dominant-Negative MMR Proteins Public Deposited

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  • The highly conserved multi-protein mismatch repair (MMR) system is known for its ability to correct post replication errors in genomic DNA. A hallmark of MMR deficiency in all organisms is microsatellite instability. The initiating proteins in the eukaryotic MMR system are hetrodimers formed with an integral MSH2 subunit and one of three other subunits; MSH3, MSH6, or MSH7. Conserved helix-turn-helix and ATPase domains in the MSH2 protein have been identified as necessary for MMR function. Mutations in these domains, followed by over-expression of the mutant proteins, have resulted in dominant-negative phenotypes in Saccharomyces cerevisiae. Described here is a project in which Arabidopsis AtMSH2 proteins carrying mutations in their helix-turn-helix and ATPase domain were constructed in “super expression” vectors then transformed into Arabidopsis thaliana with the prospect of out-competing wild-type AtMSH2 produced by the plant for hetrodimeric association with MSH3, MSH6, or MSH7. An incapacitated MMR system causes increased post-replicative mutational accumulation in cells, which can be observed through microsatellite instability. Determining the capacity of the MMR system following transformation and over-expression of the mutant AtMSH2 gene constructs is completed through detection of allele shifts in microsatellite sequences by capillary gel electrophoresis.
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