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Comparative Study
. 2008 Aug 5;105(31):10791-6.
doi: 10.1073/pnas.0801612105. Epub 2008 Jul 31.

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

Rishi H Porecha  1 , James T Stivers
Affiliations
Comparative Study

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

Rishi H Porecha et al. Proc Natl Acad Sci U S A. .

Abstract

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 approximately 10 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. Short-range sliding over the range of a helical turn allows for redundant inspection of very local DNA sequences and trapping of spontaneously emerging extrahelical uracils.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Substrate constructs used in this study. Substrates with two uracils located on the same or opposite strands of the DNA duplex are shown along with the uracil site spacings. 32P end-labeling positions are shown with asterisks, and the total length of each substrate in base pairs is given on the right.
Fig. 2.
Fig. 2.
Intramolecular transfer between uracil sites by UNG. After cleaving the glycosidic bond to either uracil in a primary excision event, UNG will either escape to bulk solution (kesc) or translocate to the second uracil (kintra) by using a sliding (kslide) or hopping (khop) pathway. The efficiency of intramolecular transfer as compared with escape is revealed by the concentration of the double-excision product on the right relative to the two single-excision products at the top and bottom of the figure.
Fig. 3.
Fig. 3.
Assay and measurement of intramolecular excision events. (A) After quenching UNG with a potent and specific oligonucleotide inhibitor, the single- and double-excision products are digested to discrete dsDNA fragments by using abasic endonuclease and a nicking restriction enzyme. (B) Time course for excision of uracils from S20. (C) Time course for excision of uracils from S400. Reactions were at 37°C with 10 mM NaCl.
Fig. 4.
Fig. 4.
Determination of intramolecular transfer probabilities (fintra). (A) Plot of fintra against time for each substrate. The curves are best fits to a second-order polynomial expression and the extrapolated value at zero time provides the true fintra. (B) fintra divided by the site excision efficiency (E) plotted as a function of uracil site spacing in nanometers. Spacings were calculated by using the wormlike chain model for the DNA polymer (23, 24). The solid curve is the best fit to Eq. 4 for hopping, and the 90% confidence intervals are plotted as dashed lines. The uracil spacings in base pairs are shown at the top of the plot for reference. The orange data point is for S56 with a NaCl concentration of 150 mM.
Fig. 5.
Fig. 5.
Determination of the uracil excision efficiency (E). (A) Schematic of pulse–chase kinetic partitioning experiment to determine the ratio of glycosidic bond cleavage (kex) to substrate dissociation (koff). Excess enzyme (1 μM) is rapidly mixed with 32P-labeled DNA substrate (20 nM) and after 2 ms of aging time the reaction is either quenched with 0.5 N HCl or chased with 60 μM unlabeled DNA trap. The ratio of bound substrate that dissociates (ST*) to that which reacts to form product (PT*) in the chase period gives the ratio kex/koff. (B) Product (squares) and substrate (triangles) concentrations during the chase time. The amount of product that is formed during the chase period must be corrected for the amount that was present after the 2-ms aging period (Pq*, dashed line); this correction is obtained from the acid-quenched samples (SI Methods). Because the trap is not 100% efficient, the released substrate can slowly rebind and react to form more product, thus the true ratio of PT*/ST* = kex/koff is obtained by extrapolation to zero time.
Fig. 6.
Fig. 6.
Short-range sliding by UNG allows trapping of extrahelical uracil bases. On landing within 10 bp of a uracil site, UNG scans the duplex with an average residence time of 0.5 ms per base pair. U/A base pairs spontaneously open with an estimated rate constant of 8 ms−1 at 37°C (see text and SI Methods). Thus, on average, UNG can sample four opening events during its binding lifetime. Binding from bulk solution to an extrahelical uracil is not a kinetically competent pathway for recognition (26).
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