A study of substrate specificity of mammalian and bacterial DNA polymerases with 5-alkyl-2′-deoxyuridine 5′-triphosphates

Abstract

DNA polymerases from procaryotic sources can utilize a variety of dTTP analogues as substrates. We studied here in vitro DNA syntheses catalyzed by DNA polymerase α and β of calf thymus, and for comparison, by the Escherichia coli DNA polymerase I large fragment enzyme in the presence of 5-alkyl derivatives of dUTP as dTTP substrate analogues, using activated DNA as template-primer. The alkyl substituents were n- alkyl (from ethyl to hexyl) and iso-alkyl (isopropyl and tert-butyl) groups. All enzymes were active in the presence of each modified dTTP, incorporation rates of [ 3H]dAMP or [ 3H]dGMP were, however, much lower with the analogues than with dTTP. According to relative incorporation rates, α-polymerase in DNA synthesis was found to be less sensitive to changes in the length of the alkyl substituent of 5-n- alkyl-dUTPs than β-polymerase or the E. coli enzyme. Evidence for the incorporation of the analogues was presented for 5-[2- 14C]isopropyl-dUTP.