Uracil DNA N-glycosylase distributively interacts with duplex polynucleotides containing repeating units of either TGGCCAAGCU or TGGCCAAGCTTGGCCAAGCU.
Uracil DNA N-glycosylase (UDG) has been used as a model enzyme to test a novel universal approach to discriminate between two possible enzymatic mechanisms of specific site location in DNA, processive (DNA-scanning mechanism) and distributive (random diffusion-mediated mechanism). Two double-stranded concatemeric polynucleotides of defined length (440-480 nucleotides) containing deoxyuridine at either every 10th or 20th nucleotide in the DNA chain were prepared by the ligation of self-complementary 10- or 20-mer oligodeoxyribonucleotides. Incubation of these polynucleotides with Escherichia coli UDG, followed by thermal breakage of the abasic sites, formed fragments that were multiples of either the 10- or the 20-mer. Since the processive and distributive mechanisms of uracil removal by UDG would be very different, the fragment distribution, generated at each time interval during the UDG reaction, should be unique. To show this, we developed a computer model illustrating both possible mechanisms of UDG functioning. The distribution of DNA fragments experimentally generated during the time course of the UDG reaction was compared with the results of the computer programs that modeled the distributive and processive mechanisms. The data indicated that uracil removal, catalyzed by UDG, is consistent with a distributive model.
Purmal
A A
AA
Department of Microbiology and Molecular Genetics, Markey Center for Molecular Genetics, University of Vermont, Burlington 05405.
Lampman
G W
GW
Pourmal
E I
EI
Melamede
R J
RJ
Wallace
S S
SS
Kow
Y W
YW
D001483:Base Sequence; D003198:Computer Simulation; D004247:DNA / Q000378:metabolism; D045647:DNA Glycosylases; D004926:Escherichia coli / Q000201:enzymology; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D011119:Polynucleotides / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D013379:Substrate Specificity; D051981:Uracil-DNA Glycosidase
D011119:Polynucleotides; D004247:DNA; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase