Abstract

BackgroundFidelity of DNA polymerases can be influenced by cation co-factors. Physiologically, Mg2+ is used as a co-factor by HIV reverse transcriptase (RT) to perform catalysis; however, alternative cations including Mn2+, Co2+, and Zn2+ can also support catalysis. Although Zn2+ supports DNA synthesis, it inhibits HIV RT by significantly modifying RT catalysis. Zn2+ is currently being investigated as a component of novel treatment options against HIV and we wanted to investigate the fidelity of RT with Zn2+.MethodsWe used PCR-based and plasmid-based alpha complementation assays as well as steady-state misinsertion and misincorporation assays to examine the fidelity of RT with Mn2+, Co2+, and Zn2+.ResultsThe fidelity of DNA synthesis by HIV-1 RT was approximately 2.5 fold greater in Zn2+ when compared to Mg2+ at cation conditions optimized for nucleotide catalysis. Consistent with this, RT extended primers with mismatched 3′ nucleotides poorly and inserted incorrect nucleotides less efficiently using Zn2+ than Mg2+. In agreement with previous literature, we observed that Mn2+ and Co2+ dramatically decreased the fidelity of RT at highly elevated concentrations (6 mM). However, surprisingly, the fidelity of HIV RT with Mn2+ and Co2+ remained similar to Mg2+ at lower concentrations that are optimal for catalysis.ConclusionThis study shows that Zn2+, at optimal extension conditions, increases the fidelity of HIV-1 RT and challenges the notion that alternative cations capable of supporting polymerase catalysis are inherently mutagenic.

Highlights

  • Fidelity of DNA polymerases can be influenced by cation co-factors

  • We show that presumed pro-mutagenic cations, such as Mn2+ and Co2+, are not mutagenic with Human immunodeficiency virus (HIV) reverse transcriptase (RT) at concentrations optimal for dNTP catalysis

  • Synthesis under the alternative divalent cations Optimal extension conditions for HIV RT with Mg2+, Mn2+, Co2+, and Zn2+ in presence of 100 μM dNTPs were determined on a 425 nt RNA template derived from the gag-pol region of the HIV genome

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Summary

Introduction

Fidelity of DNA polymerases can be influenced by cation co-factors. Mg2+ is used as a co-factor by HIV reverse transcriptase (RT) to perform catalysis; alternative cations including Mn2+, Co2 +, and Zn2+ can support catalysis. Divalent cations are essential co-factors for polymerase catalysis and are required for the RNase H activity of reverse transcriptase (RT) [1,2]. In addition to Mg2+, RT in vitro can use alternative divalent cations such as Mn2+, Cu2+, Co2+ and Zn2+ for polymerase activity [4]. These cations are important to many cellular processes and are tightly regulated. The available free concentration of all these cations is kept extremely low by cellular mechanisms [12,13]

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