The astonishingly efficient location and excision of damaged DNA bases by DNA repair glycosylases is an especially intriguing problem in biology. One example is the enzyme uracil DNA glycosylase (UNG), which captures and excises rare extrahelical uracil bases that have emerged from the DNA base stack by spontaneous base pair breathing motions. Here, we explore the efficiency and mechanism by which UNG executes intramolecular transfer and excision of two uracil sites embedded on the same or opposite DNA strands at increasing site spacings. The efficiency of intramolecular site transfer decreased from 41 to 0% as the base pair spacing between uracil sites on the same DNA strand increased from 20 to 800 bp. The mechanism of transfer is dominated by DNA hopping between landing sites of Ϸ10 bp size, over which rapid 1D scanning likely occurs. Consistent with DNA hopping, site transfer at 20-and 56-bp spacings was unaffected by whether the uracils were placed on the same or opposite strands. Thus, UNG uses hopping and 3D diffusion through bulk solution as the principal pathways for efficient patrolling of long genomic DNA sequences for damage. Shortrange sliding over the range of a helical turn allows for redundant inspection of very local DNA sequences and trapping of spontaneously emerging extrahelical uracils. facilitated diffusion ͉ search mechanism

SUMMARY The ability of fluorine in a C-F bond to act as a hydrogen bond acceptor is controversial. To test such ability in complex RNA macromolecules, we have replaced native 2′-OH groups with 2′-F and 2′-H groups in two related systems, the Tetrahymena group I ribozyme and the ΔC209 P4-P6 RNA domain. In three cases the introduced 2′-F mimics the native 2′-OH group, suggesting that the fluorine atom can accept a hydrogen bond. In each of these cases the native hydroxyl group interacts with a purine exocyclic amine. Our results give new insight about the properties of organofluorine and suggest a possible general biochemical signature for tertiary interactions between 2′-hydroxyl groups and exocyclic amino groups within RNA.

Protein enzymes establish intricate networks of interactions to bind and position substrates and catalytic groups within active sites, enabling stabilization of the chemical transition state. Crystal structures of several RNA enzymes also suggest extensive interaction networks, despite RNA's structural limitations, but there is little information on the functional and energetic properties of these inferred networks. We used double mutant cycles and pre-steady state kinetic analyses to probe the putative interaction between the exocyclic amino group of the guanosine nucleophile and the N7 atom of residue G264 of the Tetrahymena group I ribozyme. As expected, the results supported the presence of this interaction, but remarkably, the energetic penalty for introducing a CH group at the 7-position of residue G264 accumulates as the reaction proceeds towards the chemical transition state to a total of 6.2 kcal/mol. Functional tests of neighboring interactions revealed that the presence of the CH group compromises multiple contacts within the interaction network that encompass the reactive elements, apparently forcing the nucleophile to bind and attack from an altered, suboptimal orientation. The energetic consequences of this indirect disruption of neighboring interactions as the reaction proceeds demonstrate that linkage between binding interactions and catalysis hinges critically on the precise structural integrity of a network of interacting groups.

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The astonishingly efficient location and excision of damaged DNA bases by DNA repair glycosylases is an especially intriguing mechanistic problem. The problem is exemplified by the paradigm enzyme uracil DNA glycosylase (UNG) that excises rare uracil bases from genomic DNA. Here we explore the efficiency and mechanism by which UNG executes intramolecular transfer and excision of two uracil sites embedded at increasing distances from each other in duplex DNA. At low salt concentrations, the efficiency of intramolecular site excision (fintra) decreased from 61 to zero percent as the base pair spacing between uracil sites increased from 20 to 800 bp. The mechanism of transfer is dominated by one‐dimensional sliding for spacings less than about 56 bps with an estimated diffusion constant of 7 × 105 bp2/s, with predominantly three‐dimensional hopping for spacings greater than about 200 bps. At intermediate spacings, both sliding and hopping pathways are operative. Physiological salt concentration decreased fintra by 76 % at a site spacing of 56 bp, with only 1 in 10 UNG molecules cleaving the second site in an intramolecular transfer event. We develop a generally useful kinetic model for interpreting ensemble site transfer data that provides the fractional contribution of sliding and hopping pathways as a function of site spacing. Our analysis indicates that intramolecular hopping and 3D diffusion through bulk solution are the principal pathways for efficient patrolling of long genomic DNA sequences for damage. Thus, short‐range sliding events only function to allow redundant inspection of very local DNA sequences. This work was supported by NIH grant GM56834‐10 awarded to J.T.S.