Hopping enables a DNA repair glycosylase to search both strands and bypass a bound protein - PubMed (original) (raw)

Hopping enables a DNA repair glycosylase to search both strands and bypass a bound protein

Mark Hedglin et al. ACS Chem Biol. 2010.

Abstract

Spontaneous DNA damage occurs throughout the genome, requiring that DNA repair enzymes search each nucleotide every cell cycle. This search is postulated to be more efficient if the enzyme can diffuse along the DNA, but our understanding of this process is incomplete. A key distinction between mechanisms of diffusion is whether the protein maintains continuous contact (sliding) or whether it undergoes microscopic dissociation (hopping). We describe a simple chemical assay to detect the ability of a DNA modifying enzyme to hop and have applied it to human alkyladenine DNA glycosylase (AAG), a monomeric enzyme that initiates repair of alkylated and deaminated purine bases. Our results indicate that AAG uses hopping to effectively search both strands of a DNA duplex in a single binding encounter. This raised the possibility that AAG might be capable of circumnavigating blocks such as tightly bound proteins. We tested this hypothesis by binding an EcoRI endonuclease dimer between two sites of DNA damage and measuring the ability of AAG to act at both damaged sites in a single binding encounter. Remarkably, AAG bypasses this roadblock in approximately 50% of the binding events. We infer that AAG makes significant excursions from the surface of the DNA, allowing reorientation between strands and the bypass of a bound protein. This has important biological implications for the search for DNA damage because eukaryotic DNA is replete with proteins and only transiently accessible.

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Figures

Figure 1

Figure 1

Processivity assays to determine the mechanism of linear diffusion by a DNA repair glycosylase. (a) Sequences of oligonucleotides that were employed in this study. All DNA duplexes contained two εA lesions (E) on the same or opposing strands and one or two fluorescein labels (asterisk). The local sequence context for the εA lesions are marked by a solid line if they are identical for a given substrate and the 10 bp palindrome that contains a central EcoRI recognition sequence (GAATTC) is marked by a dashed line. (b) Processivity assays follow events subsequent to an initial base excision event, effectively measuring partitioning between dissociation and correlated excision at the nearby lesion site. Substrates were designed so that AAG randomly binds to and excises either of the two εA lesions to create an abasic site (Ab). AAG release is irreversible under both multiple turnover and pulse-chase conditions, because the excess substrate prevents rebinding to a released intermediate.

Figure 2

Figure 2

Ionic strength dependence for the processivity of AAG is inconsistent with a purely sliding model. Multiple turnover processivity assays were performed with either full-length (a) or Δ80 AAG (b), using substrates that contained two εA lesions separated by 25 bp (47εA2F2, ●) or 50 bp (72εA2F2, ■). The fraction processive was calculated as described in the Methods and the ionic strength dependence was fit by a cooperative model with 7 inhibitory sodium ions (5). Each data point reflects the mean value from at least two independent experiments and the error bars indicate one standard deviation (n ≥ 4). The theoretical processivity for the 72mer (□, dashed lines) was calculated from the data for the 47mer, using the sliding model described in the text (See Supporting Information).

Figure 3

Figure 3

AAG searches both strands of DNA. To test whether hopping contributes to the searching mechanism of AAG, we measured the processivity for substrates in which lesions are on the opposing strands and compared this to a substrate in which the lesions are on the same strand (47εA2F2). See Figure 1 for the DNA sequences. Multiple-turnover processivity assays were performed at an ionic strength of 200 mM for full-length AAG (blue) or 115 mM for Δ80 AAG (green). Each column represents the average of at least two independent experiments with error bars indicating one standard deviation from the mean (n ≥ 4).

Figure 4

Figure 4

Testing the effect of a protein roadblock on linear diffusion by AAG. a) The 72mer substrate is depicted with an EcoRI dimer (green) bound to the central recognition site, and with an AAG monomer (blue) bound to the abasic product from the first excision reaction. If AAG is able to bypass the tightly bound protein (dashed arrow), then processive excision of the second εA will be observed. b) Structure of the EcoRI•DNA complex (41) is from the pdb (1ERI). The surfaces of the two EcoRI monomers are shown in blue and green and the DNA is depicted as a cartoon with the backbone in orange and the central EcoRI recognition sequence in yellow. c) The structure of the complex of the catalytic domain of AAG bound to εA-DNA (42) is from the pdb (1F4R). Images were rendered with Pymol (

http://www.pymol.org

).

Figure 5

Figure 5

Effect of bound EcoRI on the processivity of AAG. Multiple turnover processivity assays were performed with 200 nM 72εA2F2, 2 nM full-length AAG, and 0, 210, and 420 nM EcoRI dimer at both 100 and 200 mM ionic strength and the calculated processivity values are shown. The presence of the tightly bound EcoRI dimer reduces the processivity of AAG by ∼50% at ionic strengths of 100 mM (black bars) and 200 mM (gray bars).

Figure 6

Figure 6

Pulse-chase processivity assays indicate that AAG can bypass a bound EcoRI dimer. a) The experimental design is depicted. Fluorescein-labeled substrate (72εA2F2) was incubated with or without EcoRI for 1 hour (t1), after which AAG was added. AAG was incubated for 40 seconds (t2), before mixing with excess unlabeled substrate (72εA2). Incubations continued for 50 minutes (t3) and aliquots were removed and analyzed by the gel-based glycosylase assay. The ratio of AAG to labeled substrate to unlabeled chase was 1:10:260. b) A representative time course for full-length AAG was performed in duplicate at an ionic strength of 100 mM. Substrate depletion is fit to a single exponential (_k_obs = _k_chem). The amplitude of ∼10% disappearance of substrate confirms that AAG was bound and excised at least one εA lesion prior to dissociation. No further glycosylase activity was observed up to 50 minutes, confirming that adequate chase was used. The build-up of products (red) and intermediates (black) were fit to single-exponentials solely to show trends. c) The fraction processive was calculated from the burst amplitudes in panel b and from additional experiments for both full-length (blue) and Δ80 AAG (green). Each column represents the average of two independent experiments with error bars representing one standard deviation from the mean (n ≥ 4). The column labeled “Control” is from reactions in which EcoRI was first bound to the unlabeled substrate instead of the labeled substrate. The final reaction conditions are identical to the +EcoRI reactions, but the endonuclease and glycosylase are bound on different DNA molecules. Therefore, the decrease in processivity is due to a direct block of AAG diffusion as opposed to an artifact of some other component of the EcoRI sample.

Scheme 1

Scheme 1

Sliding and hopping are distinct mechanisms of linear diffusion

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