Mutational analysis of arginine 276 in the leucine-loop of human uracil-DNA glycosylase

Cheng Yao Chen, Dale W. Mosbaugh, Samuel E. Bennett

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9 Citations (Scopus)

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 Arg276 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 Arg276. 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 [32P]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 Arg276 mutations did not alter this attribute.

Original languageEnglish
Pages (from-to)48177-48188
Number of pages12
JournalJournal of Biological Chemistry
Volume279
Issue number46
DOIs
Publication statusPublished - 2004 Nov 12

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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