Journal: The Journal of biological chemistry
Article Title: Mutational Analysis of Arginine 276 in the Leucine-loop of Human Uracil-DNA Glycosylase *
Figure Lengend Snippet: Ability of UNG and Arg 276 mutant proteins to form UV-catalyzed cross-links to [ 32 P]25-mer DNA A , samples (12 µl) containing 20 pmol of 5′-end 32 P-labeled oligonucleotide T-25-mer, U-25-mer, or dψU-25-mer without UNG ( lanes 1–3 , respectively) or with 40 pmol of UNG ( lanes 4–6 , respectively) were UV-irradiated for 30 min as described under “Experimental Procedures.” Following irradiation, samples were subjected to non-denaturing polyacrylamide gel electrophoresis; the gels were dried and analyzed using a PhosphorImager. The positions of the UNG × [ 32 P]DNA-25-mer cross-linked complex bands and free [ 32 P]DNA-25-mer bands are indicated by arrows . B , reaction mixtures (12 µl) were prepared in duplicate that contained 20 pmol of [ 32 P]dψU-25-mer and 40 pmol of UNG. Following UV irradiation for 0, 5, 10, 20, 30, and 45 min ( lanes 3–14 , respectively), reactions were analyzed as in A . Control reactions ( lanes 1 and 2 ), containing 20 pmol of [ 32 P]dψU-25-mer, were not irradiated. C , reaction mixtures (12 µl) were prepared in duplicate that contained 20 pmol of [ 32 P]dψU-25-mer ( closed circles ), [ 32 P]T-25-mer ( closed squares ), or [ 32 P]U-25-mer ( closed triangles ), and 40 pmol of UNG. Following UV irradiation for 0, 5, 10, 20, 30, and 45 min, reactions were analyzed as in A , and the PhosphorImager data were quantified using the ImageQuant program. The cross-linking efficiency (%) was calculated by dividing the intensity of the UNG × [ 32 P]25-mer band by the sum of the [ 32 P]25-mer and UNG × [ 32 P]25-mer bands and multiplying by 100. D , reaction mixtures (12 µl) were prepared that contained 20 pmol of [ 32 P]dψU-25-mer and 40 pmol of each Arg 276 mutant enzyme. The reactions were UV-irradiated for 10 min and analyzed as described in C . The cross-linking efficiency of each mutant preparation, indicated by the corresponding single letter amino acid abbreviation, is compared with that of UNG. Error bars represent the standard deviation of three experiments.
Article Snippet: The nucleotide sequence corresponding to the core catalytic domain of human uracil-DNA glycosylase (UNG*) was amplified in a polymerase chain reaction (100 µl) containing pUNG15 as template (0.5 µg), primers (1 µ m each) RI: 5′-GC GAATTC TTTGGAGAGAGCTGGAAG-3′, and H3: 5′-GC AAGCTT TCACAGCTCCTTCCAGTC-3′, as forward and reverse primers, respectively, 1× ThermoPol (New England Biolabs) buffer, 200 µ m each dATP, dTTP, dCTP, and dGTP, and 2 units of Deep Vent DNA polymerase.
Techniques: Mutagenesis, Labeling, Irradiation, Polyacrylamide Gel Electrophoresis, Standard Deviation