DNA translocation by human uracil DNA glycosylase: role of DNA phosphate charge

Abstract

Human DNA repair glycosylases must encounter and inspect each DNA base in the genome to discover damaged bases that may be present at a density of <1 in 10 million normal base pairs. This remarkable example of specific molecular recognition requires a reduced dimensionality search process (facilitated diffusion) that involves both hopping and sliding along the DNA chain. Despite the widely accepted importance of facilitated diffusion in protein-DNA interactions, the molecular features of DNA that influence hopping and sliding are poorly understood. Here we explore the role of the charged DNA phosphate backbone in sliding and hopping by human uracil DNA glycosylase (hUNG), which is an exemplar that efficiently locates rare uracil bases in both double-stranded DNA and single-stranded DNA. Substitution of neutral methylphosphonate groups for anionic DNA phosphate groups weakened nonspecific DNA binding affinity by 0.4-0.5 kcal/mol per substitution. In contrast, sliding of hUNG between uracil sites embedded in duplex and single-stranded DNA substrates persisted unabated when multiple methylphosphonate linkages were inserted between the sites. Thus, a continuous phosphodiester backbone negative charge is not essential for sliding over nonspecific DNA binding sites. We consider several alternative mechanisms for these results. A model consistent with previous structural and nuclear magnetic resonance dynamic results invokes the presence of open and closed conformational states of hUNG. The open state is short-lived and has weak or nonexistent interactions with the DNA backbone that are conducive for sliding, and the populated closed state has stronger interactions with the phosphate backbone. These data suggest that the fleeting sliding form of hUNG is a distinct weakly interacting state that facilitates rapid movement along the DNA chain and resembles the transition state for DNA dissociation.

Figure 1

a) The `molecular clock' approach uses a small molecule inhibitor of hUNG (uracil) to trap enzyme molecules that have hopped off the DNA chain during transfer between two uracil target sites, while leaving sliding enzymes unperturbed (9). Thus, this method allows quantitative determination of the individual contributions of hopping and sliding transfers (where the total transfer probability is P_trans = P_hop + P_slide). b) Simulations showing the dependence of facilitated transfer (P_trans) on the concentration of the trap [based on the mechanism in (a)]. As previously noted (9), the probability of locating a site by hopping includes the probability that the enzyme initially falls off the DNA (P_off) as well as the probability that it returns to the same DNA chain (P_return) without getting lost to bulk solution (P_hop = P_offP_return). The trap allows selective disruption of the hopping pathway because the probability that a hopping enzyme returns to the DNA chain decreases according to P_return = k_return/(k_reurn + k_Trap[Trap]). The utility of this approach and the numerous control experiments that confirm its utility have been previously published (9).

Figure 2

Site transfer assay. a) Schematic of the site transfer assay where a single strand of a duplex DNA substrate contains two uracil sites is reacted with hUNG ([hUNG] << [DNA]). After quenching, cleavage of the product abasic sites results in single site (AB and BC) and double site (A and C) product fragments (produced from intramolecular translocation events). Qualitatively, intramolecular translocation of hUNG is indicated by an excess of the A and C fragments under initial rate conditions (see text). b) Schematic of the reaction products as analyzed by gel electrophoresis. Bands are quantified by imaging and the intramolecular transfer efficiency is calculated from eq 1 (see text).

Figure 3

Substrate design and effects of methylphosphonate (M) substitutions on nonspecific DNA binding as measured using fluorescence anisotropy. (a) Design of 5′ fluorescein labeled oligonucleotides containing all phosphodiester or methylphosphonate (M) linkages. NS5 and NS6 and their corresponding M containing oligonucleotide sequences were chosen to match the intervening sequences in the uracil containing substrate used in the site transfer assays. (b) Equilibrium hUNG binding to oligonucleotides NS6 and NS6M. (c) Summary of determined dissociation constants for hUNG and non-specific DNA. Error bars represent mean ± 1 s.d. of at least three independent trials.

Figure 4

hUNG sliding and hopping is unaffected on double stranded DNA substrates containing intervening neutral methylphosphonate (M) linkages. a) Schematic of the substrates used (S5M, S6M). Methylphosphonate positioning was chosen so that the catalytic excision of uracil by hUNG is unaffected (22, 23). b) Gel images of the site transfer products derived from S5M in the presence and absence of uracil. c) Determination of P_trans, where the observed site transfer (P_trans^obs, eq 1) is calculated at each time point and linearly extrapolated to zero time to determine the true site transfer value (P_trans). Values at 10 and 15 mM uracil were identical indicating measurements were made within the plateau region depicted in Fig. 1b. d) Summary of the site transfer properties of hUNG for double stranded methylphosphonate containing DNA substrates compared to the all phosphodiester versions. Data for S5 and S6 were reported previously and are shown for comparison (9). Values are equal to the mean ± 1 s.d. of at least 3 trials at 0 mM Uracil and 6 at high uracil (3 each at 10 and 15 mM uracil).

Figure 5

hUNG sliding on ssDNA is unaffected by methylphosphonate substitutions. (a) Gel images of the site transfer assay in the presence and absence of uracil for S6M^ss which contains two intervening M substitutions. We note the presence of a small amount of cleavage in the zero time lane (<1%). This was found to be the result of the commercially available terminal deoxynucleotidyl transferase used in the 3′ end labeling having a very small amount of uracil DNA glycosylase activity over the >2 hr incubation at 37 °C, likely from copurification. These background bands were determined to have no effect on the site transfer calculations. (b) Linear extrapolation of P_trans^obs for S6M^ss in the presence and absence of uracil to determine P_trans. (c) Comparison of the site transfer measurements of S6M^ss to that of the all phosphodiester substrate S5^ss. Comparison with S5^ss is reasonable because site transfer by sliding on ssDNA is flat for site spacings between 5 and 10 ntds (see text).

Figure 6

Site transfer measurements of hUNG under approximated physiological ionic strength conditions (140 mM potassium glutamate, 10 mM Na-HEPES pH 7.5, 200 μM MgSO₄). (a) Gel images of the raw site transfer measurements in the presence and absence of uracil. (b) Determination of P_trans by linear extrapolation. The values are the same in the presence and absence of uracil indicating that site transfer occurs entirely by sliding under these high salt conditions.

Figure 7

DNA interactions of hUNG non-specifically bound to a destabilized thymine basepair (PDB ID: 2OXM (27), 4MF = 4-methylindole). Residues shown have a nitrogen or oxygen atom < 3.3 Å of a nitrogen atom of a DNA base or a phosphate oxygen of the DNA backbone.

Figure 8

Two-state model for hUNG sliding on nonspecific DNA. In this model hUNG exists in a highly populated “closed” recognition conformation that makes multiple interactions with the phosphate backbone as observed in crystallographic studies, and also a transient mobile “open” sliding conformation that makes little or no interactions with the phosphate backbone (this work). It is reasonable to view the open state as the aborted transition state preceding DNA dissociation. The open state, which must be present at least 5% of the total bound lifetime (see text), allows for fast movement on the DNA while also allowing time for recognition of uracil bases when they are encountered.