Uracil DNA glycosylase uses DNA hopping and short-range sliding to trap transient extrahelical uracils

Uracil incorporation within human DNA occurs at a rate of ∼2,000 events per day. The enzyme uracil DNA glycosylase (UNG) is responsible for locating these uracil bases within the immense background of undamaged DNA present in the human genome and excising them before they result in genetic changes. The mechanism by which this enzyme diffuses to uracil sites is unknown but the mechanism must reconcile two important problems in DNA damage recognition. First, the ten million-fold excess of undamaged bases relative to uracil requires that UNG not bind excessively tightly to nonspecific DNA, or the enzyme would be continuously bound to nontarget sites. Second, UNG must have a sufficient residence time at both target and nontarget basepairs such that it can detect the difference. Previous NMR dynamic studies have defined this residence time to be in the millisecond range such that UNG can trap thymine or uracil bases that emerge from the base stack due to spontaneous base pair breathing motions. These considerations put rigorous limitations on the possible mechanisms by which UNG may diffuse to its target sites and execute repair. Three-dimensional diffusion through bulk solution is not a viable mechanism because the lifetime of spontaneously extrahelical bases is too short. Therefore, intramolecular transfer mechanisms are required. Such mechanisms could involve three-dimensional hopping or one-dimensional sliding along the DNA chain.;These mechanisms of diffusion may be distinguished by employing a series of DNA substrates that contain two uracil sites spaced by a defined number of base pairs. The probability of an enzyme excising uracil at one site and then successfully transferring to the second site without dissociation to bulk solution decreases as the site spacing increases. Moreover, the dependence of the transfer probability as a function of the site spacing is different for hopping and sliding mechanisms, allowing these processes to be experimentally distinguished.;This thesis describes the synthesis and characterization of DNA substrates containing two uracils at defined site spacings and the kinetic experiments and analytical approaches to elucidate the intramolecular transfer mechanism of UNG. The results reveal that UNG performs intramolecular transfer by iterative hopping between DNA landing sites. During each landing event UNG then checks each base pair over a length of DNA consisting of about one helical turn. Dissociation to bulk solution is a frequent event during this search process as the intramolecular transfer efficiency is only about 40% at a site spacing of only 20 base pairs. This enzymatic mechanism of hopping, followed by careful inspection of each base pair over a very local region of DNA, elegantly balances the need to encounter many genomic sites within a short period of time with the requirement to remain in contact with the DNA long enough to inspect spontaneously emerging bases.