Molecular mechanisms responsible for mutation during DNA replication and during the repair of DNA damage will be examined using bacteriophage T7 infected Escherichia coli. The DNA product from in vitro DNA replication, DNA repair, and recombination systems that closely mimic in vivo T7 DNA metabolism will be encapsulated with a DNA packaging system so as to reveal the genetic consequences of the prior biochemical manipulations. This system will be used to study mechanisms responsible for genetic deletions between short homologous DNA sequences. To measure the frequency of deletion events an insertion of synthetically made DNA will be introduced into a natural T7 DNA substrate so that the insertion is bracketed by direct repeats. This insertion will interrupt the coding sequence of the (non-essential) gene for T7 ligase. Perfect excision of the insertion between the direct repeats restores a coding sequence that allows the phage to synthesize ligase. A systematic study of how various parameters of the interruptive DNA sequence affect the frequency with which it is deleted from the genome will be made. The roles of recombination and replication fork slip-page in the deletion events will be determined. Factors that cause extensive deletions and those that promote one base pair frameshifts will be compared. An effort will be made to identify both phage and host gene products important in deletion formation. A search will be made for T7 mutation strains that cause an abnormally high frequency of either frameshifts or deletions. Finally, the effects of some types of DNA damage on frameshift and deletion formation will be considered. This research should improve understanding of how a natural duplex DNA molecule is coupled with high accuracy and what factors compromise the fidelity of replication. The long term goal is to provide basic knowledge that will allow more meaningful judgments and more accurate extrapolations to be made concerning health hazards caused by genetic damage inflicted by environmental insults.
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