2′‐Deoxyribonucleoside Phosphoramidate Triphosphate Analogues as Alternative Substrates for E. coli Polymerase III

Abstract

Thermostable bacterial polymerases like Taq, Therminator and Vent exo(-) are able to perform DNA synthesis by using modified DNA precursors, a property that is exploited in several therapeutic and biotechnological applications. Viral polymerases are also known to accept modified substrates, and this has proven crucial in the development of antiviral therapies. However, non-thermostable polymerases of bacterial origin, or engineered variants, that have similar substrate tolerance and could be used for synthetic biology purposes remain to be identified. We have identified the α subunit of Escherichia coli polymerase III (Pol III α) as a bacterial polymerase that is able to recognise and process as substrates several pyrophosphate-modified dATP analogues in place of its natural substrate dATP for template-directed DNA synthesis. A number of dATP analogues featuring a modified pyrophosphate group were able to serve as substrates during enzymatic DNA synthesis by Pol III α. Features such as the presence of potentially chelating chemical groups and the size and spatial flexibility of the chemical structure seem to be of major importance for the modified leaving group to play its role during the enzymatic reaction. In addition, we could establish that if the pyrophosphate group is altered, deoxynucleotide incorporation proceeds with an efficiency varying with the nature of the nucleobase. Our results represent a great step towards the achievement of a system of artificial DNA synthesis hosted by E. coli and involving the use of altered nucleotide precursors for nucleic acid synthesis.

Base‐Modified Nucleic Acids as a Powerful Tool for Synthetic Biology and Biotechnology

“…The primer‐template duplex had seven overhanging dC residues at the 5′‐end of the template. We examined two thermophilic DNA polymerases, namely, Taq DNA polymerase (Taq) and Vent (exo−) DNA polymerase (Vent (exo−)), as well as two mesophilic E. coli DNA polymerases, namely, the Klenow fragment of DNA polymerase I (KF (exo−)) and the α‐subunit of DNA polymerase III (PolIIIα) (Table S1 in the Supporting Information) . It is well documented that the presence of Mn 2+ ions can support the incorporation of modified nucleoside triphosphates because of a reduction of substrate specificity .…”

“…[21] Enzymatic reactions (10 mL) were performed in either 1X ThermoPol buffer (New England Biolabs) for reactions with thermophilic polymerases (Taq and Vent (exoÀ)), or 1X NEBuffer 2( New England Biolabs) for KF (exoÀ), or 1X PolIII buffer (20 mm Tris-HCl, pH 7.5, 1mm MgCl 2 ,1 0mm DTT,2 0mgL À1 BSA, 4% glycerol) for reactions with PolIIIa.T he final reaction mixtures contained radiolabeled primer-template duplex (P1:T7, 50 nm), DNA polymerases (Taq, Vent (exoÀ), KF (exoÀ)( 25 UmL À1 ) and PolIIIa (1200 UmL À1 )), different dGxTP (100 mm,w here Gx is G 1 -G 6 ), and with or without MnCl 2 (1 mm). [21] Enzymatic reactions (10 mL) were performed in either 1X ThermoPol buffer (New England Biolabs) for reactions with thermophilic polymerases (Taq and Vent (exoÀ)), or 1X NEBuffer 2( New England Biolabs) for KF (exoÀ), or 1X PolIII buffer (20 mm Tris-HCl, pH 7.5, 1mm MgCl 2 ,1 0mm DTT,2 0mgL À1 BSA, 4% glycerol) for reactions with PolIIIa.T he final reaction mixtures contained radiolabeled primer-template duplex (P1:T7, 50 nm), DNA polymerases (Taq, Vent (exoÀ), KF (exoÀ)( 25 UmL À1 ) and PolIIIa (1200 UmL À1 )), different dGxTP (100 mm,w here Gx is G 1 -G 6 ), and with or without MnCl 2 (1 mm).…”

Isoguanine and 5‐Methyl‐Isocytosine Bases, In Vitro and In Vivo

“…Vent ( exo -), Taq and KF ( exo -) polymerases were purchased from New England Biolabs, Pfu ( exo -) from Agilent Technologies, Tfi ( exo -) from Life technologies, T7 sequenase from Affymetrix, T4( exo -) from Lucigen and Pol III α-subunit was expressed and purified as described. [ 32 ]…”

Enzymatic Incorporation of Modified Purine Nucleotides in DNA

“…The DNA polymerases (EC 2.7.7.7 for all) used in the incorporation experiments were Taq, Vent ( exo ‐), and Klenow Fragment ( exo ‐) (New England Biolabs); Tfi (Invitrogen); Pfu ( exo ‐) (Agilent Technologies); T4 ( exo ‐) (Lucigen); Sequenase Version 2.0 (Affymetrix); and Pol III α‐subunit (expression and purification are described by Giraut et al …”