Construction of Fluorescently-Tagged and Adenosine Nucleotide-Binding Mutations of the Human MutS Homolog Heterodimer MSH2-MSH3

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The Ohio State University

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DNA mismatch repair (MMR) is a highly conserved system for correcting mispaired nucleotides arising from misincorporation errors during DNA replication, genetic recombination, and chemical or physical damage. The MutS homologues (MSH) and MutL homologues (MLH/PMS) are the fundamental components of MMR and are conserved from bacteria to humans. The MSH proteins initiate MMR via mismatch legion recognition. One human MSH complex in particular, hMSH2-hMSH3, recognizes small insertion deletion loops (IDL) and repetitive DNA sequences. Inherited mutations in many MMR genes including hMSH2 lead to a predisposition for colorectal cancer (hereditary non-polyposis colorectal cancer, HNPCC). Also, the hMSH2-hMSH3 complex has been implicated in the expansion of tri-nucleotide repeats in disorders such as Huntington’s disease and myotonic dystrophy. The role hMSH2-hMSH3 plays in this expansion remains enigmatic. Two mutations made to the Walker A nucleotide binding domain of hMSH2 and hMSH3 will allow for a detailed study of the mechanics of this complex in recognizing and binding DNA lesions, as well as the signaling of downstream MMR components. A detailed study of the conformational changes the protein undergoes in lesion recognition will also be possible via fluorescently tagged MSH2 and MSH3 subunits for use in fluorescence resonance energy transfer.



MutS, MSH3, Mismatch Repair, MMR