DNTP binding

Stereoview of the polymerase active site of HIV-1 RT [38]. The amino acid residues that compose the putative dNTP-binding site, including the three catalytically essential aspartic acids, are shown with side chains. The double-stranded nucleic acid is shown with the atomic model in the HIV-1 RT/DNA/Fab complex. The dNTP-binding site consists of structural elements from both protein and nucleic acid. The precise composition, position, and conformation of the template-primer can affect the recognition of... 

Structural analysis of HIV-l RT has shown that most of the NRTI-resistance mutations are not located close to the putative dNTP-binding site and are unlikely to have a direct impact on the binding of dNTP analogs (Figure 5 and... 

A close-up view showing the relative locations of the commonly identified drug-resistance mutations for NRTIs (in dark-gray) and for NNRTIs (in light-gray) with respect to the bound DNA. Most of the NRTI-resistance mutations are not located at the putative dNTP-binding site, but are at positions to have potential interactions with the nucleic acid template-primer. Conversely, all the NNRTI-re si stance mutations are clustered around the NNIBP and have direct contacts with NNRTIs or have direct effect on... 

Although most polymerases conform to the general kinetic scheme, some polymerases have different mechanisms with regard to p/t binding and selection. Other aspects in the polymerase cycle such as dNTP binding, chemistry, and the conformational change will be discussed in later sections. 

The pol / p/t ddCTP structure shows that, in the absence of a downstream DNA fragment, the lyase domain does not interact with the DNA and is positioned some distance from the active site (Pelletier et al, 1994). However, in a structure solved with gapped DNA, the lyase domain binds to the 5 -phosphate in the DNA gap and interacts with its own carboxy-terminus in the thumb (Sawaya et al, 1997). In common with all polymerases, the fingers subdomain closes down around the correct nucleotide and its complementary template base. This motion corresponds to a 30-degree rotation of a-helix N (the dNTP-binding site) around oc-helix M and thus brings helix N and its bound nucleotide into position to probe correct W-C base pairing. 

Fig. 4.1.2. A. Structure of Thermits aquaticus (Taq) DNA polymerase bound to the DNA primer-template complex. B. "Open and closed conformation after dNTP binding of Taq DNA polymerase. Structures were built on PDB entries 3KTQ and 4KTQ. 

Figure 13.4 Polymerase bypass of bulky lesions, (a) Chemical structure of B[o]P-dC. The dNTP binding site (yellow circle) of the model replicative polymerase BF is blocked by the B[fl]P-dC adduct (red circle, labeled [BPJdC) (PDB 1XC9). Dpo4 can flip the B[o] P-dC adduct (red circle) out of the polymerase active site, which allows the incoming dNTP to bind (PDB 2IA6). (b) Y-family...

Polymerases Undergo a Global Conformational Change upon dNTP Binding 352... 

Figure 4 Simplified kinetic scheme of single-nucleotide incorporation by a DNA polymerase. Step 1, Mg-dNTP binding step 2, N-subdomain closing step 3, catalytic Mg + binding step 4, nucleotidyl transfer (chemistry) step 5, catalytic Mg " dissociation step 6, N-subdomain reopening step 7, pyrophosphate release. E = DNA polymerase in open conformation E = closed conformation D = DNA substrate D + = DNA product elongated by addition of one nucleotide N = Mg dNTP M = catalytic Mg " P = Mg PP. Reproduced with permission from M. Bakhtina S. Lee Y. Wang C. Dunlap B. Lamarche M. D. Tsai, Biochemistry 2005, 44, 5177-5187. Copyright 2005 American Chemical Society.

Figure 10 Superimposition of rapid chemical quench (open circles O) and stopped-flow fluorescence (blue) assays. In (a) tryptophan emission was detected, and in (b) the fluorescence change from 2-AP was monitored. Insets show the dNTP binding-induced conformational change In the presence of dideoxy-terminated DNA substrate. Adapted with permission from A. K. Showalter B. J. Lamarche M. Bakhtina M. I. Su K. H. Tang M. D. Tsai, Chem. Rev. 2006, 106, 340-360. Copyright 2006 American Chemical Society.

Previous stopped-flow fluorescence assays investigating matched dNTP incorporation showed that both the fast and the slow fluorescence transitions demonstrated a hyperbolic dependence on dNTP concentra-tion. " " " Similarly, the dNTP dependence of both the fast and the slow fluorescence phases during mismatched dNTP incorporation in stopped-flow has been examined. The observed rate constants for the fast and the slow phases, individually plotted as a function of dNTP concentration, reveal that both phases demonstrate a hyperbolic dependence on dNTP concentration (parameters obtained for k2, K, k o, and d,app as described in Section 8.10.4.2.3 and reported in Table 1). The observed hyperbolic dependence of the fast phase on mismatched dNTP largely indicates that this phase originates from a conformational change induced by mismatched dNTP binding. 

In contrast to DNA pol substrate analogs, non-nucleotide inhibitors (NNI) of DNA-dependent DNA polymerases are potentially able to specifically interact with different regions on DNA pol surface. They can be competitive in relation to the dNTP binding site (in this case we can talk about nucleotide mimics), bind to DNA template binding area and directly prevent initial DNA interaction, have allosteric binding site and, thus, exhibit non-competitive type of inhibition. 

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