Alternative divalent cations (Zn²⁺, Co²⁺, and Mn²⁺) are not mutagenic at conditions optimal for HIV-1 reverse transcriptase activity.
BACKGROUND
Fidelity of DNA polymerases can be influenced by cation co-factors. Physiologically, Mg(2+) is used as a co-factor by HIV reverse transcriptase (RT) to perform catalysis; however, alternative cations including Mn(2+), Co(2+), and Zn(2+) can also support catalysis. Although Zn(2+) supports DNA synthesis, it inhibits HIV RT by significantly modifying RT catalysis. Zn(2+) is currently being investigated as a component of novel treatment options against HIV and we wanted to investigate the fidelity of RT with Zn(2+).


METHODS
We 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 Mn(2+), Co(2+), and Zn(2+).


RESULTS
The fidelity of DNA synthesis by HIV-1 RT was approximately 2.5 fold greater in Zn(2+) when compared to Mg(2+) 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 Zn(2+) than Mg(2+). In agreement with previous literature, we observed that Mn(2+) and Co(2+) dramatically decreased the fidelity of RT at highly elevated concentrations (6 mM). However, surprisingly, the fidelity of HIV RT with Mn(2+) and Co(2+) remained similar to Mg(2+) at lower concentrations that are optimal for catalysis.


CONCLUSIONS
This study shows that Zn(2+), 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.
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, 20742, USA. vasuachu@umd.edu.
Vasudevan
V
Achuthan
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, 20742, USA. jdestefa@umd.edu.
Jeffrey J
JJ
DeStefano
D000595:Amino Acid Sequence; D001483:Base Sequence; D002413:Cations, Divalent / Q000494:pharmacology*; D003035:Cobalt / Q000494:pharmacology; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D007763:Lac Operon / Q000235:genetics; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000187:drug effects; D009154:Mutation; D015032:Zinc / Q000494:pharmacology
D002413:Cations, Divalent; D003035:Cobalt; D008345:Manganese; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D015032:Zinc