Mutational analysis of arginine 276 in the leucine-loop of human uracil-DNA glycosylase.
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.
Chen
Cheng-Yao
CY
Molecular and Cellular Biology Program, Department of Environmental and Molecular Toxicology, and the Environmental Health Sciences Center, Oregon State University, Corvallis, Oregon 97331-7301, USA.
Mosbaugh
Dale W
DW
Bennett
Samuel E
SE
D001120:Arginine / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D045647:DNA Glycosylases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004260:DNA Repair; D006801:Humans; D007930:Leucine / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011485:Protein Binding; D011487:Protein Conformation; D051981:Uracil-DNA Glycosidase; D014764:Viral Proteins / Q000378:metabolism
D014764:Viral Proteins; C027619:uracil-DNA glycosylase inhibitor protein, B. subtilis bacteriophage; D004247:DNA; D001120:Arginine; D045647:DNA Glycosylases; D051981:Uracil-DNA Glycosidase; D007930:Leucine