Investigation of the stability of D5SIC-DNAM-incorporated DNA duplex in Taq polymerase binary system: a systematic classical MD approach

Abstract

DNA polymerases are fundamental enzymes that play a crucial role in processing DNA with high fidelity and accuracy ensuring the faithful transmission of genetic information. The recognition of unnatural base pairs (UBPs) by polymerases, enabling their replication, represents a significant and groundbreaking discovery with profound implications for genetic expansion. Romesberg et al. examined the impact of DNA containing 2,6-dimethyl-2H-isoquiniline-1-thione: D5SIC (DS) and 2-methoxy-3-methylnaphthalene: DNAM (DN) UBPs bound to T. aquaticus DNA polymerase (Taq) through crystal structure analysis. Here, we have used polarizable and nonpolarizable classical molecular dynamics (MD) simulations to investigate the structural aspects and stability of Taq in complex with a DNA duplex including a DS-DN pair in the terminal 3' and 5' positions. Our results suggest that the flexibility of UBP-incorporated DNA in the terminal position is arrested by the polymerase, thus preventing fraying and mispairing. Our investigation also reveals that the UBP remains in an intercalated conformation inside the active site, exhibiting two distinct orientations in agreement with experimental findings. Our analysis pinpoints particular residues responsible for favorable interactions with the UBP, with some relying on van der Waals interactions while other on Coulombic forces.

Fig. 1

Binary complex of Taq with the artificial base pair DS–DN where (A) DS is externally intercalated, EXT_SYN (left) and EXT_ANTI (right) (B) DS is internally placed with respect to DN; INT_SYN (left) and INT_ANTI (right).

Fig. 2

UB–NB distances obtained from (A) AMOEBA and (B) AMBER force field-mediated simulations. In crystal structure, DS–DC and DN–DG is 6.69 Å and 4.64 Å respectively for EXT_SYN and 4.98 Å and 6.04 Å respectively for INT_SYN.

Fig. 3

3D representations of average RMSF values of the protein–DNA residues and associated RMSF plots of three replicates of EXT and INT structures; (A) SYN and (B) ANTI conformers obtained from AMBER simulations.

Fig. 4

Conformational change through Intra- and Inter-strand flipping observed during the MD simulations. (Figure taken from preprint with permission).

Fig. 5

O–S distance for INT_SYN and INT_ANTI conformers obtained from AMBER simulation. O–S distance of crystal structure of INTSYN is 7.4 Å. Distance value are in Å.

Fig. 6

3D representation of non-covalent interactions between polymerase residues and UBP through EDA for Ext_SYN. Residues with Coulomb interactions < −35.0 kcal mol⁻¹ and vdW interactions < −2.5 kcal mol⁻¹ are highlighted. Energy values are in kcal mol⁻¹.

Fig. 7

Non-covalent interactions between polymerase residues and for Int_SYN. Residues with Coulomb interactions < −35.0 kcal mol⁻¹ and interactions < −2.5 kcal mol⁻¹ are highlighted. Energy values are in kcal mol⁻¹.

Fig. 8

Difference in interaction profiles (ΔE_Coul and ΔE_vdW) between EXT_SYN and INT_SYN. Energy values are in kcal mol⁻¹.

Fig. 9

Difference in interaction profiles (ΔE_Coul and ΔE_vdW) between (A) EXT_SYN and EXT_ANTI, (B) (A) INT_SYN and INT_ANTI. Energy values are in kcal mol⁻¹.