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

Dmitry G Semizarov,Andrey A Arzumanov,Natalya B Dyatkina,Albert Meyer,Sophie Vichier-Guerre,Gilles Gosselin,Bernard Rayner,Jean-Louis Imbach,Alexander A Krayevsky

doi:10.1074/jbc.272.14.9556

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 alpha, beta, and epsilon from human placenta and reverse transcriptases of human immunodeficiency virus and avian myeloblastosis virus incorporate into the DNA chain only natural beta-D-dNTPs, whereas calf thymus terminal deoxynucleotidyl transferase incorporates two nucleotide residues of alpha-D-dNTP and extends the resulting oligonucleotide in the presence of beta-D-dNTPs. The latter enzyme also extended alpha-anomeric D-oligodeoxynucleotide primers in the presence of beta-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.

Highlights

It has been shown [3] that 2Ј,3Ј-dideoxy-␤-Lthymidine 5Ј-triphosphate and 2Ј,3Ј-dideoxy-2Ј,3Ј-didehydro-␤L-thymidine 5Ј-triphosphate are incorporated into DNA chains by HIV reverse transcriptase, E. coli DNA polymerase I, and T7 DNA polymerase, but their affinity to the HIV enzyme is 10 –50 times lower than that of their ␤-D-isomers
The fidelity of DNA synthesis catalyzed by HIV reverse transcriptase is rather low (24 –26), and the probability of incorrect dNMP incorporation is higher for the template positions remote from the primer 3Ј terminus by more than two nucleotide residues
Similar results were obtained for avian myeloblastosis virus reverse transcriptase

Summary

Introduction

It has been shown [3] that 2Ј,3Ј-dideoxy-␤-Lthymidine 5Ј-triphosphate and 2Ј,3Ј-dideoxy-2Ј,3Ј-didehydro-␤L-thymidine 5Ј-triphosphate are incorporated into DNA chains by HIV reverse transcriptase, E. coli DNA polymerase I, and T7 DNA polymerase, but their affinity to the HIV enzyme is 10 –50 times lower than that of their ␤-D-isomers. One of the reasons for that is the similarity in substrate specificity between TDT and some other DNA polymerases, especially DNA polymerase ␤ [11, 12] and endogeneous reverse transcriptases [13]. We have recently found [4] that dNTP analogs with trans-like orientation of the nucleic base and triphosphate residue efficiently and selectively inhibit DNA synthesis catalyzed by TDT. In this paper we synthesized all four possible stereoisomers of dNTPs with respect to the C-1Ј and C-4Ј carbon atoms and evaluated them as substrates for template-dependent DNA polymerases ␣, ␤, and ⑀ from human placenta, reverse transcriptases from HIV and avian myeloblastosis virus, and TDT from calf thymus. The retention times for the corresponding ␤-D-dNTPs are given in parentheses

Methods

Results

Conclusion