The reproduction of all forms of life depends on the accurate replication of the genome, which is facilitated by DNA polymerases.1
These enzymes increase the rate of phosphodiester bond formation by many orders of magnitude compared to the corresponding reaction in water.2
The rate of incorporation of an incoming wrong nucleotide (W) is drastically slower than the corresponding rate of the right nucleotide (R) for DNA polymerases that exhibit high replication fidelity (for reviews see refs.3-6
). Thus it is important to quantify the factors that control the fidelity.
Despite significant experimental progress in studies of the fidelity of various DNA polymerases (e.g., refs.3-10
), we still do not have a clear quantitative picture of the energetics of this process. A significant progress has been made in recent theoretical studies, which explored the contributions to fidelity from the binding and chemical steps,11-18
as well as on the possible roles of conformational changes,16,19,20
and protein motions.21
However, the progress in these key areas is still at a relatively early stage.
Further insight about the fidelity issue has been provided in a recent study,21
which used computer simulations to model the effect of mutations on the structure of Pol β, and examined the corresponding electrostatic effects by a qualitative analysis. Additionally, theoretical studies of related problems have been reported recently.22-25
Molecular dynamics simulations have helped to delineate a possible sequence of conformational change events after dNTP binding in the catalytic cycle of Pol β.19,20,26
Simulation studies also enabled to test various catalytic proposals of nucleotide transfer reactions.12,27
Finally, an attempt to progress in a more quantitative direction in the comparison of calculated and observed mutational effects has been reported.28
This study also introduced a useful approach of constructing interaction matrices and using them in probing the transfer of information between the binding and catalytic sites.
One of the intriguing aspects of the action of DNA polymerases is the role of substrate-induced conformational changes in the catalytic process and the influence of these changes on DNA replication fidelity. This aspect is related to the general issue of the role of the enzyme conformational landscape in catalysis29-31
and to proposals that dynamical effects and coupled motions play a major role in enzyme catalysis.32,33
Although careful studies (for review, see refs.34,35
) have questioned the validity of such proposals, there are aspects that were not explored in a conclusive way and one of these aspects is the relationship between DNA polymerase conformational changes and replication fidelity, that will be explored in this work. More specifically, based on structural and kinetic studies of Pol β, it is generally believed that the first step of the nucleotide insertion pathway includes the N–subdomain’s (equivalent to the fingers subdomain of right-handed DNA polymerases) closing triggered by correct dNTP binding, while binding of an incorrect dNTP will hamper this closing, consistent with an “induced fit” mechanism.36
It has been suggested that conformational changes that occur prior to the chemical step play a major role in establishing the fidelity of Pol β (e.g., ref.37
) and DNA polymerases in general.4-6
Instead, we believe that it is considerably more likely that fidelity is determined by the binding energy plus the activation barrier of the chemical step, and that the barrier for conformational changes between the open and closed forms is not likely to determine the fidelity in Pol β.14,15
Experimental evidence also indicates that the open to closed subdomain transition is too rapid to be rate determining.38-41
A recent computational study16
has attempted to generate the catalytic landscape for Pol β and obtained insightful conclusions including capturing some of the structural changes, that were not known at the time of that work (see discussion). However, after the crucial elucidation of the structure of R (ref.42
) and W (ref.43
) we can move to a more concrete ground. In fact, the advances in structural studies led to several interesting theoretical studies.17,44,45
However, some of these studies17,27,44,46,47
involve some questionable conclusions. For example, a QM/MM study on the prechemistry barrier, before the nucleotide transfer reaction, in human DNA pol β with a G:A mismatch in the active site44
was estimated to be about 14 kcal/mol. Based on this, the authors suggested that the free energy required for formation of the prechemistry state is the major contributing factor to the decrease in the rate of incorrect nucleotide incorporation compared with correct nucleotide insertion and therefore to the fidelity enhancement. Also in a more recent QM/MM study on DNA polymerase IV (Dpo4),48
it was argued that the prechemistry reorganization of the catalytic site ( which brings the enzyme to an active conformation from its x-ray position prior to the chemical reaction) would cost approximately 4.0 to 9.0 kcal/mol. However, both these studies employed a problematic treatment, using basically energy minimization without proper relaxation and sampling.49
Such treatments can lead to artificial barriers as they will be illustrated in the present work.
In a complex enzymatic reaction, involving the association of the enzyme with more than one substrate, it is unlikely that the highest energy barrier will be approached in a single step from the ground state reactants. Instead there will be one or more intermediate steps along the path way. This fact and other considerations led different workers46,50
to suggest that the intermediate points can serve as kinetic checkpoints. Basically it was argued that although the presence of such checkpoints will not change the overall fidelity of the reaction , they will define the pathway by which that fidelity would be realized. In the polymerase reaction, the intermediate steps or checkpoints will allow rejection of inappropriately paired dNTPs before the polymerase attempts phosphodiester bond formation of such substrates.50
Unfrotunetly, these concepts are not based on clear molecular concepts and have not been supported by consistent simulations. In fact, even the common suggestion that fidelity is due to the induced fit effect, is not based on clear structure-energy analysis.16
( see also discussion in section IV. 2)
Fortunately, with the new available structural information42,43
we are entering a stage where one can apply consistent theoretical analysis and establish the molecular origin of replication fidelity, while analyzing (and disproving if needed) problematic proposals. Such an analysis is the purpose of the present work.
In order to set the stage for our discussion and analysis we start with the general working hypothesis of . This conceptual figure has emerged from our previous studies of DNA polymerases15,16
where we concluded (based on calculations of the barrier for the rate determining chemical step) that the path along the coordinate for the chemical process pass at a somewhat different value of the “orthogonal” coordinate of the protein structural changes (typically considered as the motion between the open and closed forms). We would like to emphasize that, this view is fundamentally different than related assumptions19
that the barriers for the conformational changes between the open and closed form contribute to fidelity or to the reaction rate. The barriers for the motion from the open to closed form in the reactant state of R (from rORS(R)
in ) is not likely to influence fidelity, or for that matter, the reaction rate for both W and R, as long as they are lower than the chemical barriers.35
Similarly the interesting possibility that the pre-chemistry barriers are different for W and R (e.g., ref.19
) is not likely to influence fidelity as long as those barriers are lower than the chemical barrier. In other words, we are focusing here on the path between the bound incoming dNTP to the product (rCRS(R)
) and on its dependence on the protein conformation, assuming (based on experimental findings) that the chemical step is rate-determining (this point will be further considered in the Discussion section IV).
Figure 1 Schematic free energy surfaces that illustrate possible coupling between the conformational and chemical coordinates for the insertion of (A) correct and (B) incorrect dNTP by a DNA polymerase. rO, and rC, designate respectively the open and closed configurations. (more ...)
With the above perspective in mind we try to move forward from our previous study (using now the actual structures of R and W of Pol β) and address the following questions: (i) the possible relationship between the chemical barrier for R and W and the orthogonal protein conformational coordinate; (ii) The general relationship between the landscape of the protein conformations and the catalytic power of enzymes; (iii) the molecular meaning of the “induced fit” effect and its relationship, if any, to fidelity; (iv) the problems with the prechemistry concept; and (v) the idea of coupled motions and check points.