This study provides the first characterization of a DNA repair enzyme dynamically inspecting undamaged DNA. The data clearly reveal millisecond time scale motions of UNG that are induced upon binding to nontarget DNA. Since the induced dynamic behavior is localized to residues known to be involved in nontarget DNA binding and target searching (6
), the motions are likely to be functionally relevant to the scanning and target search steps of DNA repair as depicted in . Although NMR is a powerful method for detecting dynamic motions of biomolecules, the measurements by themselves do not reveal the structural changes that are being sampled. Thus, other experimental observations must be utilized to link the observed dynamics to structure and the process of searching and recognizing DNA damage.
The regions of UNG that show induced dynamics upon binding to nontarget DNA reflect a subset of regions that undergo a clamping movement upon binding to both nontarget and target DNA (6
). The relative displacements in the backbone amide positions of UNG at early and late points along the reaction coordinate are shown in a. The early displacements, which are relatively large, involve transitioning from the free enzyme to the exosite complex with nontarget DNA (blue line, a). The late displacements, which are very small, reflect structural changes that occur upon transitioning from the exosite complex to the specific uracil complex (green line, a). Thus, the enzyme has largely assumed its catalytically active closed conformation at the exosite complex even though the flipped base is only one-sixth of the way along the 160° rotation leading to the active site. The inference is that the observed dynamic fluctuations of the DNA bound enzyme reflects exchange between a predominantly closed conformation as observed in the exosite crystal structure and another bound state that is intermediate between the free state and the exosite complex. Such fluctuations on the millisecond time scale are consistent with the estimated millisecond life time of UNG at individual base pairs as it stochastically slides along the DNA contour (7
). Oscillations between a looser binding state and a closed state that allows sampling of base pair opening events provide a plausible mechanism for scanning and pausing along the DNA contour to inspect the duplex for uracil bases (1
Figure 4. 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 (more ...)
If an open to closed conformational transition is responsible for the observed dynamic behavior of the DNA-bound enzyme, then it would be expected that the equilibrium scaffold of UNG would possess low-energy motions that allow this type of reversible clamping motion. To investigate this possibility, we performed a normal mode analysis (NMA) of UNG using the crystallographic coordinates from the exosite complex (pdb code 2OXM) (30
). The lowest frequency mode obtained from this analysis resulted in atom displacements that recapitulate those observed in moving from the free enzyme to the exosite complex (dashed line, a). The two extrema from this lowest frequency mode are shown in b with the exosite DNA duplex included for reference. A video of the complete trajectory is available as Supplementary Video 1
online. The mechanism of short-range DNA scanning that is suggested by this analysis involves oscillation of the enzyme between an open state that allows stochastic, thermally driven translocation along the DNA strand and a closed state where the pincer and finger regions interact more intimately with the DNA major and minor grooves (b). The closed state seen in the exosite complex is especially well poised for the rapid capture of thymine and uracil bases that spontaneously emerge from the DNA base stack (6
A surprising observation is that UNG is not dynamic until it binds to nontarget DNA. This property distinguishes UNG from several previously studied enzyme systems where pre-existing and catalytically competent dynamic motions were detected in the free enzyme (10–14
). The induced flexibility of the UNG backbone upon DNA binding is distinct from several transcription factors that are highly dynamic in the free state and assume a rigid conformation only upon binding to their cognate sequences (31
). Given the high concentration of nonspecific DNA binding sites in the cell nucleus and UNG's micromolar affinity for nontarget DNA (a), the enzyme would be expected to be bound to DNA at all times in vivo
. This environment would select for dynamic properties of the enzyme–DNA complex that optimize efficient repair rather than properties of the free enzyme which may be under different selection pressures.
A functional model for the role of nontarget DNA binding in the function of UNG is summarized in . The absence of significant dynamics in the free enzyme indicates that free UNG inhabits a narrow conformational energy well and is unable to sample conformations that resemble the bound state. Thus, the free energy of DNA binding is used to populate unstable states that are inaccessible in the absence of DNA, and it is only upon binding to nontarget DNA that dynamic modes relevant to the search process become activated. The bound states of UNG are most reasonably assigned to a weakly interacting open conformation that is competent for sliding along the DNA contour and resembles the free enzyme. The second closed state resembles the early exosite structure and is poised to detect extrahelical bases. The closed state may also possess additional dynamic motions in the sub-millisecond regime that allow efficient capture of transiently emerging bases. These findings reveal how the free energy of DNA binding can be used to modulate the conformational and dynamic landscape of an enzyme allowing it to scan the DNA contour and respond to intrinsic base pair dynamics.
Figure 5. 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 (more ...)