The long range goal of this project is to understand the biochemical basis for the exchange and maintenance of genetic information in Escherichia coli. To help achieve this objective, the experiments outlined in this proposal will focus on three enzymes which have been implicated in genetic recombination, DNA repair, and spontaneous mutagenesis. These proteins are: exonuclease I (sbcB); exonuclease V (recB and recC); and DNA helicase II (uvrD). The structural genes for all three enzymes have been cloned and will be used to purify large quantities of each protein for detailed analysis of their individual catalytic activities and their interactions with other nucleic acid enzymes. Particular attention will be given to determine if there is a third subunit of exonuclease V, since it has now been shown that 97,000 dalton polypeptide is encoded between the recB and recC loci. Additionally, a series of conditionally lethal recC mutants will be studied both genetically and biochemically. Mutationally altered forms of DNA helicase II will be cloned, purified, and characterized in order to examine the role of this protein in DNA repair synthesis and spontaneous mutagenesis. Finally, a variety of exonuclease I mutations have been cloned and will be used to determine why some only suppress certain of the phenotypic properties associated with exonuclease V deficiency.
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