2 Results and Discussion 19
2.5 Elongation of modified nucleotide analogs
2.5.3 Increase in incorporation efficiency of the alkyne modified substrates by using a
2.5.3 Increase in incorporation efficiency of the alkyne modified substrates by using a mutated KlenTaq variant
The present structural data indicates that during enzymatic introduction of modified substrates the enzyme DNA interactions on the primer site are clearly affected. The idea suggests itself that to strengthen the interaction framework on the template site might compensate the decreased in stabilizing the primer Figure 53 Zoom into the active site of KlenTaqddC-‐alkyne (brown). (A) Close-‐up view of the complexation of Mg2+ ions by the incoming ddCalkyneTP and the amino acids crucial for catalysis. (B) Octahedral coordination of the metal ion A in KlenTaqddC-‐alkyne. (C) Coordination of metal ion A by five ligands in KlenTaq1QTM (green). (D) Superimposition of KlenTaqddC-‐alkyne and KlenTaq1QTM showing the same orientation as in (A).
strand. Keeping this in mind, the acceptance of the alkyne modified substrates was further investigated by a previously evolved KlenTaq I614K, M747K mutant (henceforth termed KlenTaqDM) (181). This variant combines two single mutants derived from directed-‐evolution approaches, which are known for its improved translesion synthesis capability. It was found that the mutation of a single nonpolar amino acid side chain by a cationic side chain increases the processivity of KlenTaqDM by expanded positively charged surface potential areas. The mutations are located at the template site, thus KlenTaqDM was used to prove the above mentioned hyphosesis. The competition experiments catalyzed by KlenTaqDM result in a slightly improved incorporation efficiency for dTalkyneTP showing an approximately 5-‐fold lower incorporation efficiency compared to the natural counterparts (Figure 54). In correlation with this results the competition experiment carried out with dCalkyneTP vs. dCTP show similar trends. Surprisingly dCalkyneTP seems to be slightly better accepted by KlenTaqDM than the natural counterpart. These findings support the idea of the balance between the enzyme substrate interactions.
Figure 54 Acceptance of dTalkyneTP and dCalkyneTP by KlenTaqDM DNA polymerase. (A) Competition experiments of dTalkyneTP versus dTTP.
PAGE analysis of an exemplary competition experiments employing KlenTaqDM DNA polymerase (reaction time: 1 min). The ratio of dTalkyneTP/dTTP was varied from 1/1 to 100/1 (1/1, 2/1, 4/1, 10/1, 20/1, 50/1, 100/1). Lane P: 5’-‐32P-‐labeled primer; lane C: only dTTP (ratio: 0/1); lane Talkyne: only dTalkyneTP (ratio: 1/0). The product bands were quatified to evaluate the incorporation efficiencies of dTalkyneTP (■, dashed line) and dTTP (•, solid line) catalyzed by KlenTaq. The conversion in % was plotted versus the concentration. A zoom into the cross spot indicates the approximate ratio where both nucleotides are equally incorporated with a dotted line. (B) Same as in (A) using dCalkyneTP and dCTP instead. The ratio of dCalkyneTP/dCTP was varied from 1/10 to 10/1 (1/10, 1/4, 1/2, 1/1, 2/1, 4/1, 10/1).
2.5.4 Discussion
KlenTaq DNA polymerase – a member of the A family – is a high fidelity DNA polymerase, known for its accuracy in DNA synthesis. However, the obtained structures of KlenTaq capturing suitable alkyne modified substrates in the active site illustrate once more the plasticity of the enzyme. The model of
“active site tightness” described by Kool relies on a sterically defined binding pocket of the incoming nucleotide (42). Since the natural substrates also differ from each other, there are also relative flexible regions in this binding pocket. This fact underpins the idea that the enzyme is able to respond to the incoming nucleotide and shows certain flexibility at these positions. Attaching the modification at the C5 atom of the pyrimidines has two major advantages. Firstly, modifications at this position cause minor disruption of the DNA duplex, since the modification points to the major groove. Secondly, it also results in minor disruption of the ternary complex of the enzyme as the present structural study emphasizes. The disturbances of the enzyme substrates interactions are minimal, since the enzyme is sensitive to alterations at that position. Moreover, the present structural study indicates that DNA polymerases are not only able to accept modifications, they can interact and stabilize the modified substrates. The alkyne linked ethynylphenyl ring of dTalkyneTP and dCalkyneTP is able to interact with positively charged amino acid side chains like arginine or lysine via cation-‐π interaction. Similar effects were found in KlenTaq processing a dendron modified nucleotide dTdendTP (176). In this case the Arg660 interacts with the nitrogen of the propargylamide linkage and the 3’-‐primer terminus, contributing to increase the incorporation efficiency. Whereas a spinlabel modified nucleotide dTspinTP is examined as a disruption and Arg660 is released from its stabilizing interaction to the primer strand and flips out to make room for the spinlabel (176). In correlation with these findings, the well acceptance of dTalkyneTP and dCalkyneTP of might lead back to the cation-‐π interaction to Arg587 and Lys663. However, the contribution of Lys663 to stabilize the modified substrates via cation-‐π interaction remains to be elucidated. Given that Lys663 exhibits an enlarged distance from the amino group to the center of the aromatic ring compared to Arg587. The introduction of the modification at the C5 atom of the nucleobase does not induce a conformational change of Lys663, whereas it clearly affects the conformation of Arg587. Similar orientations of Arg587 are found in KlenTaq processing a guanosine (PDB: 1QSS), or when purines are incorporated opposite an abasic site (PDB: 3LWL / 3RR8 /3RRG). During bypass of an abasic site Arg587 was identified as a stabilizing factor for the incoming purine nucleotides via hydrogen bond and/or cation-‐π interactions (149). Further, functional in combination with mutational studies of DNA polymerase I from E. coli (Klenow Fragment), sharing a high degree of structural and sequence similarity with KlenTaq, identified Arg682, which is homolog to Arg587 in KlenTaq, as base substitution mutator (163). The R682A variant of Klenow Fragment shows a decreased fidelity at the insertion step, underpinning the hypothesis that interactions to Arg587 contribute to the substrate acceptance. This might also be transferable to modified substrates. Thus, in addition to the previously identified Arg660 as a stabilizing factor for propargylamide linked modifications (176), the present structural and functional study identified Arg587 as a stabilizing guide for an ethynylphenylethynyl linkage.
Further the introduction of two consecutive alkyne modified substrates revealed that the benzene rings can communicate with each other via π-‐π stacking interaction. This interaction dissolves the conjugated aromatic system, reaching over nucleobase and modification, forcing the nucleotide analog to twist the
modification in respect to the nucleobase. Thereby, the disruption of the enzyme’s active site is minimized, indicated by the reorientation of Arg660 and Arg587. The presence of amino acid with a long side chain near the substrates such as arginine clearly contributes to the enzyme plasticity and flexibility.
The results suggest that modifications, which require a rigid linkage, might be better tolerated by a DNA polymerase if they contain an aromatic ring, which can communicate with the protein or with each other via cation-‐π or π-‐π interaction, respectively. In fact, several examples of dNTPs that are processed by DNA polymerases and modified via a C5 ethynylphenylethynyl linker have been reported (136, 182). For instance, Burgess and coworkers demonstrated that rigid conjugated linkers are able to enhance the spectroscopic properties of dye labeled nucleotide analogs (182). Further the incorporation studies of these nucleotides showed that the acceptance by AmpliTaqFS DNA polymerase (widely applied in high throughput DNA sequencing methods) is increased in line with the linker length. The dye labeled nucleotide with the ethynylphenylethynyl linkage is recognized by TaqFS, whereas the use of the dye labeled nucleotide analog with a simple alkyne linkage in primer extension reactions result in no significant product formation. These insights and the present structural study suggest that implementing an aromatic ring at the discussed position in modified dNTPs may improve their substrate properties. This enhancement of design guidelines for the development of new modified dNTPs, in combination with directed evolution of DNA polymerases (178-‐180), will stimulate the development of future applications.
For instance, the employment of KlenTaqDM – an evolved KlenTaq mutant – showing enhanced translesion synthesis capability, results in slightly increased incorporation efficiencies for dTalkyneMP and dCalkyneMP, underscoring the impact of evolved DNA polymerases.