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Characterization of Escherichia coli...

Sung, Jung-Suk.

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

Record Type:	Electronic resources : Monograph/item
Title/Author:	Characterization of Escherichia coli double-strand uracil-DNA glycosylase and analysis of uracil-initiated base excision DNA repair./
Author:	Sung, Jung-Suk.
Description:	395 p.
Notes:	Source: Dissertation Abstracts International, Volume: 63-08, Section: B, page: 3567.
Contained By:	Dissertation Abstracts International63-08B.
Subject:	Biology, Genetics. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3061921
ISBN:	049377758X

Characterization of Escherichia coli double-strand uracil-DNA glycosylase and analysis of uracil-initiated base excision DNA repair.
Sung, Jung-Suk.

Characterization of Escherichia coli double-strand uracil-DNA glycosylase and analysis of uracil-initiated base excision DNA repair. - 395 p.

Source: Dissertation Abstracts International, Volume: 63-08, Section: B, page: 3567.

Thesis (Ph.D.)--Oregon State University, 2003.

<italic>Escherichia coli</italic> double-strand uracil-DNA glycosylase (Dug) was purified to apparent homogeneity from bacteria that were defective in uracil-DNA glycosylase (Ung). After cloning the <italic>dug</italic> 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 <italic> E. coli</italic> 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 <italic> E. coli</italic> (<italic>ung</italic>) cell-free extracts. The relative rate of repair of uracil- and ethenocytosine-containing DNA in isogenic <italic> E. coli</italic> 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 <italic>E. coli</italic> cell-free extracts that were proficient or deficient in Ung activity and determined to be 5.5 × 10<super>−4 </super> and 19.7 × 10<super>−4</super>, respectively.

ISBN: 049377758XSubjects--Topical Terms:

1017730
Biology, Genetics.

Characterization of Escherichia coli double-strand uracil-DNA glycosylase and analysis of uracil-initiated base excision DNA repair.
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245 1 0 $a Characterization of Escherichia coli double-strand uracil-DNA glycosylase and analysis of uracil-initiated base excision DNA repair.
300 $a 395 p.
500 $a Source: Dissertation Abstracts International, Volume: 63-08, Section: B, page: 3567.
500 $a Adviser: Dale W. Mosbaugh.
502 $a Thesis (Ph.D.)--Oregon State University, 2003.
520 $a <italic>Escherichia coli</italic> double-strand uracil-DNA glycosylase (Dug) was purified to apparent homogeneity from bacteria that were defective in uracil-DNA glycosylase (Ung). After cloning the <italic>dug</italic> 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 <italic> E. coli</italic> 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 <italic> E. coli</italic> (<italic>ung</italic>) cell-free extracts. The relative rate of repair of uracil- and ethenocytosine-containing DNA in isogenic <italic> E. coli</italic> 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 <italic>E. coli</italic> cell-free extracts that were proficient or deficient in Ung activity and determined to be 5.5 × 10<super>−4 </super> and 19.7 × 10<super>−4</super>, respectively.
590 $a School code: 0172.
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650 4 $a Biology, Molecular. $3 1017719
650 4 $a Chemistry, Biochemistry. $3 1017722
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