A rapid reaction analysis of uracil DNA glycosylase indicates an active mechanism of base flipping

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Abstract
Uracil DNA glycosylase (UNG) is the primary enzyme for the removal of uracil from the genome of many organisms. A key question is how the enzyme is able to scan large quantities of DNA in search of aberrant uracil residues. Central to this is the mechanism by which it flips the target nucleotide out of the DNA helix and into the enzyme-active site. Both active and passive mechanisms have been proposed. Here, we report a rapid kinetic analysis using two fluorescent chromophores to temporally resolve DNA binding and base-flipping with DNA substrates of different sequences. This study demonstrates the importance of the protein–DNA interface in the search process and indicates an active mechanism by which UNG glycosylase searches for uracil residues.
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