Stereoisomers of Deoxynucleoside 5′-Triphosphates as Substrates for Template-dependent and -independent DNA Polymerases

Abstract

All four possible stereoisomers of dNTP with regard to deoxyribofuranose C-1′ and C-4′ carbon atoms were studied as substrates for several template-dependent DNA polymerases and template-independent terminal deoxynucleotidyl transferase. It was shown that DNA polymerases α, β, and ϵ from human placenta and reverse transcriptases of human immunodeficiency virus and avian myeloblastosis virus incorporate into the DNA chain only natural β-D-dNTPs, whereas calf thymus terminal deoxynucleotidyl transferase incorporates two nucleotide residues of α-D-dNTP and extends the resulting oligonucleotide in the presence of β-D-dNTPs. The latter enzyme also extended α-anomeric D-oligodeoxynucleotide primers in the presence of β-D-dNTPs. None of the studied enzymes utilized L-dNTPs. These data indicate that template-dependent DNA polymerases are highly stereospecific with regard to dNTPs, whereas template-independent terminal deoxynucleotidyl transferase shows less stereodifferentiation. It is likely that the active center of the latter enzyme forms no specific contacts with the nucleic bases of both nucleotide substrate and oligonucleotide primer. All four possible stereoisomers of dNTP with regard to deoxyribofuranose C-1′ and C-4′ carbon atoms were studied as substrates for several template-dependent DNA polymerases and template-independent terminal deoxynucleotidyl transferase. It was shown that DNA polymerases α, β, and ϵ from human placenta and reverse transcriptases of human immunodeficiency virus and avian myeloblastosis virus incorporate into the DNA chain only natural β-D-dNTPs, whereas calf thymus terminal deoxynucleotidyl transferase incorporates two nucleotide residues of α-D-dNTP and extends the resulting oligonucleotide in the presence of β-D-dNTPs. The latter enzyme also extended α-anomeric D-oligodeoxynucleotide primers in the presence of β-D-dNTPs. None of the studied enzymes utilized L-dNTPs. These data indicate that template-dependent DNA polymerases are highly stereospecific with regard to dNTPs, whereas template-independent terminal deoxynucleotidyl transferase shows less stereodifferentiation. It is likely that the active center of the latter enzyme forms no specific contacts with the nucleic bases of both nucleotide substrate and oligonucleotide primer.