Mutations in which nucleotides are added or deleted from the DNA sequence play a major role in pathology. Microsatellite instability signals the susceptibility of cells to frameshift mutations of critical importance in tumorigenesis. Expansion of triplet repeats sets off the pathogenesis of a set of neuromuscular diseases. The innate stability of the DNA is determined by its sequence but the maintenance of sequence is ensured by the surveillance activities of a set of proteins. We propose: a) to determine the relative importance of structural factors in DNA that lead to slippage by systematically measuring mutations in repeat nucleotide runs of different length and base composition. For this purpose we plan to construct a substrate in which any target sequence can be placed in a particular position to set a reporter gene either in- or out-of-frame; b) We propose to test the hypothesis that the dnaE and dnaN subunits of the replicative Escherichia coli DNA polymerase reduce slippage by anchoring (repeated) DNA nucleotides to particular amino acids in protein; c) Eukaryotic mismatch repair proteins (mutS homologs) are specialized for the correction of base substitution or slippage errors. We will determine if the bacterial mutS protein can distinguish between the two types of errors by selecting for mismatch repair mutants correcting primarily one or the other; d) Proofreading activity requires association of the proofreading exonuclease with the polymerase subunit. We will determine whether proofreading results from modification of the exonuclease or from an altered interaction with other subunit domains. Our goal is to learn by mutational analysis the location of the amino acid sites in proteins which recognize and interact with repeated sites in DNA to prevent or control slippage. We suppose that this understanding will lead to intervention strategies to control slippage in individuals deficient in particular stages of the surveillance process.
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