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 (LTNG2) was overproduced in E. coli
cells and purified to apparent homogeneity. While UNG2 retained ~9 % of UNG
activity, it did form a stable complex with Ugi. Paradoxically, low concentrations of
NaC1 and MgC1₂ 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. / Graduation date: 2005
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/30026 |
| Date | 09 December 2004 |
| Creators | Chen, Cheng-Yao |
| Contributors | Schimerlik, Michael I. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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