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Biochemical characterization and mut...

Chen, Cheng-Yao.

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Biochemical characterization and mutational analysis of human uracil-DNA glycosylase.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Biochemical characterization and mutational analysis of human uracil-DNA glycosylase./
Author:	Chen, Cheng-Yao.
Description:	263 p.
Notes:	Source: Dissertation Abstracts International, Volume: 66-02, Section: B, page: 0872.
Contained By:	Dissertation Abstracts International66-02B.
Subject:	Chemistry, Biochemistry. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3165839
ISBN:	0542011433

Biochemical characterization and mutational analysis of human uracil-DNA glycosylase.
Chen, Cheng-Yao.

Biochemical characterization and mutational analysis of human uracil-DNA glycosylase. - 263 p.

Source: Dissertation Abstracts International, Volume: 66-02, Section: B, page: 0872.

Thesis (Ph.D.)--Oregon State University, 2005.

PCR-based codon-specific random mutagenesis and site-specific mutagenesis were performed to construct a library of 18 amino acid changes at Arg276 in the conserved leucine-loop of the core catalytic domain of human uracil-DNA glycosylase (UNG). Each Arg276 mutant was then overproduced in E. coli cells and purified to apparent homogeneity by conventional chromatography. All of the R276 mutant proteins formed a stable complex with the uracil-DNA glycosylase inhibitor protein (Ugi) in vitro, suggesting that the active site structure of the mutant enzymes was not perturbed. The catalytic activity of all mutant proteins was reduced; the least active mutant, R276E, exhibited 0.6% of wild-type UNG activity, whereas the most active mutant, R276H, exhibited 43%. Equilibrium binding measurements utilizing a 2-aminopurine-deoxypseudouridine DNA substrate showed that all mutant proteins displayed greatly reduced base flipping/DNA binding. However, the efficiency of UV-catalyzed cross-linking of the R276 mutants to single-stranded DNA was much less compromised. Using a concatemeric [32P]U•A DNA polynucleotide substrate to assess enzyme processivity, UNG was shown to use a processive search mechanism to locate successive uracil residues, and Arg276 mutations did not alter this attribute. A transient kinetics approach was used to study six different amino acid substitutions at Arg276 (R276C, R276E, R276H, R276L, R276W, and R276Y). When reacted with double-stranded uracil-DNA, these mutations resulted in a significant reduction in the rate of base flipping and enzyme conformational change, and in catalytic activity. However, these mutational effects were not observed when the mutant proteins were reacted with single-stranded uracil-DNA. Thus, mutations at Arg276 effectively transformed the enzyme into a single-strand-specific uracil-DNA glycosylase. The nuclear form of human uracil-DNA glycosylase (UNG2) was overproduced in E. coli cells and purified to apparent homogeneity. While UNG2 retained &sim;9% of UNG activity, it did form a stable complex with Ugi. Paradoxically, low concentrations of NaCl and MgCl2 stimulated UNG2 catalytic activity as well as the rate of rapid fluorescence quenching; however, the rate of uracil flipping was reduced. When UNG2 bound pseudouracil-containing DNA, conformational change was not detected.

ISBN: 0542011433Subjects--Topical Terms:

1017722
Chemistry, Biochemistry.

Biochemical characterization and mutational analysis of human uracil-DNA glycosylase.
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500 $a Adviser: Michael I. Schimerlik.
502 $a Thesis (Ph.D.)--Oregon State University, 2005.
520 $a PCR-based codon-specific random mutagenesis and site-specific mutagenesis were performed to construct a library of 18 amino acid changes at Arg276 in the conserved leucine-loop of the core catalytic domain of human uracil-DNA glycosylase (UNG). Each Arg276 mutant was then overproduced in E. coli cells and purified to apparent homogeneity by conventional chromatography. All of the R276 mutant proteins formed a stable complex with the uracil-DNA glycosylase inhibitor protein (Ugi) in vitro, suggesting that the active site structure of the mutant enzymes was not perturbed. The catalytic activity of all mutant proteins was reduced; the least active mutant, R276E, exhibited 0.6% of wild-type UNG activity, whereas the most active mutant, R276H, exhibited 43%. Equilibrium binding measurements utilizing a 2-aminopurine-deoxypseudouridine DNA substrate showed that all mutant proteins displayed greatly reduced base flipping/DNA binding. However, the efficiency of UV-catalyzed cross-linking of the R276 mutants to single-stranded DNA was much less compromised. Using a concatemeric [32P]U•A DNA polynucleotide substrate to assess enzyme processivity, UNG was shown to use a processive search mechanism to locate successive uracil residues, and Arg276 mutations did not alter this attribute. A transient kinetics approach was used to study six different amino acid substitutions at Arg276 (R276C, R276E, R276H, R276L, R276W, and R276Y). When reacted with double-stranded uracil-DNA, these mutations resulted in a significant reduction in the rate of base flipping and enzyme conformational change, and in catalytic activity. However, these mutational effects were not observed when the mutant proteins were reacted with single-stranded uracil-DNA. Thus, mutations at Arg276 effectively transformed the enzyme into a single-strand-specific uracil-DNA glycosylase. The nuclear form of human uracil-DNA glycosylase (UNG2) was overproduced in E. coli cells and purified to apparent homogeneity. While UNG2 retained &sim;9% of UNG activity, it did form a stable complex with Ugi. Paradoxically, low concentrations of NaCl and MgCl2 stimulated UNG2 catalytic activity as well as the rate of rapid fluorescence quenching; however, the rate of uracil flipping was reduced. When UNG2 bound pseudouracil-containing DNA, conformational change was not detected.
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