Characterization of Escherichia coli double-strand uracil-DNA glycosylase and analysis of uracil-initiated base excision DNA repair Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/vx021h60q

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  • Escherichia coli double-strand uracil-DNA glycosylase (Dug) was purified to apparent homogeneity from bacteria that were defective in uracil-DNA glycosylase (Ung). After cloning the dug gene, recombinant Dug was overexpressed, purified, and characterized with respect to activity, substrate specificity, product DNA binding, and mechanism of action. Purified Dug excised both uracil and ethenocytosine specifically from double-stranded DNA substrates. One distinctive characteristic of Dug was that the purified enzyme removed a near stoichiometric amount of uracil from DNA containing U/G mispairs. The observed lack of turnover was attributed to tight binding of Dug to the apyrimidinic-site (AP) contained in the DNA reaction product. Catalytic activity was stimulated in the presence of E. coli endonuclease IV that caused AP-site incision and dissociation of Dug. By using enzyme complementation experiments, Dug was shown to initiate uracil-initiated base excision repair (BER) in E. coli (ung) cell-free extracts. The relative rate of repair of uracil- and ethenocytosine-containing DNA in isogenic E. coli cells that were proficient or deficient in Ung and/or Dug was measured using a novel competition assay. Complete ethenocytosine-initiated BER displayed an absolute requirement for Dug and occurred at the same rate as uracil-initiated BER in the presence of both Ung and Dug. However, the rate of Dug-mediated ethenocytosine-DNA repair was 8-fold faster than that of uracil-DNA mediated by Dug. The distribution of BER patch sizes associated with both uracil- and ethenocytosine-containing DNA showed similar results. In both cases, DNA repair synthesis utilized predominantly a long patch BER mechanism involving the incorporation of 2-20 nucleotides. A previously unidentified "very long patch" mechanism of BER involving the incorporation of more than 200 nucleotides was identified and shown to be mediated by DNA polymerase I. The rate-limiting step associated with uracil-initiated BER was found to involve DNA ligase and the distribution of BER patch size was modulated by the ratio of DNA polymerase I and DNA ligase. The fidelity of DNA repair synthesis associated with complete uracil-DNA BER was measured using E. coli cell-free extracts that were proficient or deficient in Ung activity and determined to be 5.5 x 10⁻⁴ and 19.7 x 10⁻⁴, respectively.
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