Abstract
Uracil residues are eliminated from cellular DNA by uracil-DNA glycosylase, which cleaves the N-glycosylic bond between the uracil base and deoxyribose to initiate the uracil-DNA base excision repair pathway. Co-crystal structures of the core catalytic domain of human uracil-DNA glycosylase in complex with uracil-containing DNA suggested that arginine 276 in the highly conserved leucine intercalation loop may be important to enzyme interactions with DNA. To investigate further the role of Arg(276) in enzyme-DNA interactions, PCR-based codon-specific random mutagenesis, and site-specific mutagenesis were performed to construct a library of 18 amino acid changes at Arg(276). All of the R276X mutant proteins formed a stable complex with the uracil-DNA glycosylase inhibitor protein in vitro, indicating that the active site structure of the mutant enzymes was not perturbed. The catalytic activity of the R276X preparations was reduced; the least active mutant, R276E, exhibited 0.6% of wildtype activity, whereas the most active mutant, R276H, exhibited 43%. Equilibrium binding studies utilizing a 2-aminopurine deoxypseudouridine DNA substrate showed that all R276X mutants displayed greatly reduced base flipping/DNA binding. However, the efficiency of UV-catalyzed cross-linking of the R276X mutants to single-stranded DNA was much less compromised. Using a concatemeric [(32)P]U.A DNA polynucleotide substrate to assess enzyme processivity, human uracil-DNA glycosylase was shown to use a processive search mechanism to locate successive uracil residues, and Arg(276) mutations did not alter this attribute.
Highlights
The uracil-DNA glycosylase family-1 enzymes (UDGs)1 are named for their homology to the first uracil-DNA glycosylase activity observed, Escherichia coli uracil-DNA glycosylase (Ung), described by Lindahl et al [1]
Overproduction and Purification of uracil-DNA glycosylase (UNG) and R276X Proteins—Codon-specific random mutagenesis was performed on arginine 276 of the human UNG gene and 18 R276X (R276X) mutants were isolated by activity screening as described under “Experimental Procedures.”
To facilitate the mutational analysis of human uracil-DNA glycosylase, the recombinant core catalytic domain of human uracil-DNA glycosylase (UNG*), consisting of residues 85–304 (numbering according to mitochondrial pre-form UNG1 [16]), UNG* containing six N-terminal histidine residues, designated UNG, and the R276X mutant UNG proteins were overproduced in E. coli and purified as described under “Experimental Procedures.”
Summary
The uracil-DNA glycosylase family-1 enzymes (UDGs)1 are named for their homology to the first uracil-DNA glycosylase activity observed, Escherichia coli uracil-DNA glycosylase (Ung), described by Lindahl et al [1].
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