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������ and 19.7 x 10������, respectively. / Graduation date: 2003
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/31840 |
| Date | 04 June 2002 |
| Creators | Sung, Jung-Suk |
| Contributors | Mosbaugh, Dale W. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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