Exploration of factors driving incorporation of unnatural dNTPS into DNA by Klenow fragment (DNA polymerase I) and DNA polymerase  

Abstract

In order to further understand how DNA polymerases discriminate against incorrect dNTPs, we synthesized two sets of dNTP analogues and tested them as substrates for DNA polymerase α (pol α) and Klenow fragment (exo−) of DNA polymerase I (Escherichia coli). One set of analogues was designed to test the importance of the electronic nature of the base. The bases consisted of a benzimidazole ring with one or two exocyclic substituent(s) that are either electron-donating (methyl and methoxy) or electron-withdrawing (trifluoromethyl and dinitro). Both pol α and Klenow fragment exhibit a remarkable inability to discriminate against these analogues as compared to their ability to discriminate against incorrect natural dNTPs. Neither polymerase shows any distinct electronic or steric preferences for analogue incorporation. The other set of analogues, designed to examine the importance of hydrophobicity in dNTP incorporation, consists of a set of four regioisomers of trifluoromethyl benzimidazole. Whereas pol α and Klenow fragment exhibited minimal discrimination against the 5- and 6-regioisomers, they discriminated much more effectively against the 4- and 7-regioisomers. Since all four of these analogues will have similar hydrophobicity and stacking ability, these data indicate that hydrophobicity and stacking ability alone cannot account for the inability of pol α and Klenow fragment to discriminate against unnatural bases. After incorporation, however, both sets of analogues were not efficiently elongated. These results suggest that factors other than hydrophobicity, sterics and electronics govern the incorporation of dNTPs into DNA by pol α and Klenow fragment.

Human DNA Polymerase α Uses a Combination of Positive and Negative Selectivity To Polymerize Purine dNTPs with High Fidelity

“…Since many of the analogues can form a WatsonCrick hydrogen bond(s) with T and, therefore, can be considered as dATP analogues, we defined wrong as polymerization opposite A, C, and G. Figure 8 shows that the ability of pol α to discriminate against polymerization of the analogues does not closely correlate with hydrophobicity, consistent with the results of previous studies (11). While the more hydrophobic bases tend to give dNTPs with lower fidelity, there are multiple exceptions to this trend.…”

“…The relatively non-specific incorporation of dNTPs lacking N-1 and N-3 indicates that after these dNTPs bind, the enzyme can readily adopt a catalytically active conformation, even though the bases of the template and incoming dNTP cannot form a correctly shaped base-pair (For example, the relatively rapid polymerization of 5,6-dinitrobenzimidazole, 5-nitroindole, or 4-methylbenzimidazole opposite A and G ( (11,12) and Table 2, respectively)). Upon adding back either N-1 or N-3 to the unnatural base, pol α adopts a catalytically active conformation much less readily.…”

“…Indeed, these enzymes misincorporate the natural NTPs orders of magnitude faster than they will incorporate NTPs containing unnatural bases incapable of Watson-Crick hydrogen bonding with the template, a situation diametrically opposed to that for pol α. Klenow fragment, an A family polymerase, has been suggested to use shape as the primary determinant for fidelity (8)(9)(10)(40)(41)(42). However, various groups have also shown that KF will also efficiently incorporate bases whose shape does not resemble a canonical base (11,12,(43)(44)(45)(46).…”

“…Thus, a correctly shaped base-pair is not essential for dNTP polymerization by pol α. These studies also indicated that the rapid polymerization of these unnatural, low fidelity bases did not result from their relatively high hydrophobicity (11).…”

“…, and 9-β-D-2′-deoxyribofuranosyl-(3-deazaguanine)-5′-triphosphate (d3DGTP) were synthesized as previously described (9,11,(16)(17)(18)(19). Triphosphorylation of 9-(2-deoxy-β-D-erythropentofuranosyl)-1-deazaadenine to 9-β-D-2′-deoxyribofuranosyl-(1-deazaadenine)-5′-triphosphate (d1DATP) was performed by Sierra Bioresearch (Tuscon, AZ).…”