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Results: 5

1.
Figure 4.

Figure 4. From: Nontarget DNA binding shapes the dynamic landscape for enzymatic recognition of DNA damage.

Backbone atom displacements of UNG upon DNA binding and lowest frequency NMA of the enzyme. (a) Comparison between amide nitrogen displacement in the lowest frequency normal mode of the exosite DNA complex (black dashed line) and the observed amide displacements between free UNG (pdb code 2OXM) and the exosite complex (pdb code 1AKZ) (blue line). (b) The two extrema of the lowest frequency normal mode of UNG indicate an open to closed conformational transition. A video of the atom displacements in this normal mode trajectory is available as Supplementary Video 1.

Joshua I. Friedman, et al. Nucleic Acids Res. 2009 June;37(11):3493-3500.
2.
Figure 5.

Figure 5. From: Nontarget DNA binding shapes the dynamic landscape for enzymatic recognition of DNA damage.

The free energy of nontarget DNA binding is used to alter the dynamic landscape of UNG. Free UNG (pdb code 1AKZ) populates a single open conformation within a steep energy well. The free energy of DNA binding is used to destabilize regions of the UNG structure and lower the activation barrier for UNG to sample an open and closed state in the search complex (see text). The free energy differences between the free enzyme and the complex are drawn arbitrarily.

Joshua I. Friedman, et al. Nucleic Acids Res. 2009 June;37(11):3493-3500.
3.
Figure 3.

Figure 3. From: Nontarget DNA binding shapes the dynamic landscape for enzymatic recognition of DNA damage.

Nontarget DNA binding to human UNG. (a) Fluorescence anisotropy measurements were used to determine the dissociation constant of UNG for a 5′-fluorescein-labeled 10-mer duplex DNA of the sequence 5′ TCGATCGATG 3′. (b) The crystal structure of human UNG bound to nontarget DNA (yellow) showing the central thymidine in the transient exosite specific for T and U (Figure 1) (6). (c) The same view of UNG as panel (b) but the heteronuclear (1H–15N) weighted chemical shift perturbations of its backbone amides upon addition of nontarget DNA are color coded onto the surface. The DNA molecule is removed for clarity.

Joshua I. Friedman, et al. Nucleic Acids Res. 2009 June;37(11):3493-3500.
4.
Figure 1.

Figure 1. From: Nontarget DNA binding shapes the dynamic landscape for enzymatic recognition of DNA damage.

The uracil target search mechanism of human UNG. The search process begins by diffusion to a nontarget site in DNA followed by short-range scanning of the DNA helix. During a short scanning event lasting about 5 ms, UNG can trap thymine or uracil bases (X) in a specific exosite binding pocket as they spontaneously emerge from the DNA base stack with estimated rates of 8 ms−1 at 37°C (7). A molecular sieving mechanism is used to reject the methyl group of thymine and allow uracil (U) to selectively proceed to the enzyme active site. The scanning and target discrimination step is probed by the NMR experiments in this work.

Joshua I. Friedman, et al. Nucleic Acids Res. 2009 June;37(11):3493-3500.
5.
Figure 2.

Figure 2. From: Nontarget DNA binding shapes the dynamic landscape for enzymatic recognition of DNA damage.

Dynamics of free UNG and its complex with nontarget DNA. (a) Global fits of the relaxation dispersion profiles of the amide nitrogens of the UNG–DNA complex (green). Selected residues of the UNG–DNA complex were used to globally optimize a single exchange rate constant [kex, Equation (3)]. All residues are located in close proximity to the DNA binding pocket of UNG. With the exception of Ser169, horizontal lines are drawn through the data for free UNG (blue) to emphasize the νcpmg field independent behavior for resonances of the free enzyme. (b) Dynamic regions that interact with the DNA strand containing the extrahelical thymine in the exosite (PDB 2OXM). The locations of the backbone amides used in the global fitting in (a) are colored red in this view. The extrahelical thymine is shown in ball and stick representation and the other DNA strand is omitted for clarity. (c) Global view of the dynamics and chemical shift perturbations induced by nontarget DNA binding. All backbone amides in UNG that show significant exchange in the DNA-bound state are colored red. The width of the backbone of UNG is drawn proportional to the amide chemical shift perturbation brought about by nontarget DNA binding (Figure 3c). The magnitudes of the chemical shift changes between free and bound UNG are not well correlated with the exchange contribution to the line widths in the bound state indicating that exchange involves two bound states. The bound DNA is shown in cyan looking down the helical axis.

Joshua I. Friedman, et al. Nucleic Acids Res. 2009 June;37(11):3493-3500.

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