Human DNA polymerases alpha and beta are able to incorporate anti-HIV deoxynucleotides into DNA.

Abstract:

Deoxynucleoside analogs, AZT and/or ddN, are the therapeutic agents currently utilized to inhibit the human immunodeficiency virus (HIV) reverse transcriptase. The effects of their anabolic products, AZT-triphosphate (AZT-TP) and ddCTP on human cellular DNA metabolic processes were studied using highly purified, structurally and enzymatically defined forms of the two major human host DNA polymerases, alpha and beta, and compared to those of the reverse transcriptase purified from HIV viron. Human DNA polymerase alpha during processive DNA synthesis is able to incorporate AZT-monophosphate (AZT-MP) but not ddCMP into DNA, causing chain termination. During its initial encounter with a primer terminus, polymerase alpha is able to incorporate both AZT-MP and ddCMP into DNA chains. Polymerase beta is able to incorporate AZT-MP and ddCMP into DNA, causing chain termination in both modes of DNA synthesis. Steady state kinetic analyses demonstrate that polymerase alpha inserts one AZT-MP molecule into DNA for every 2500 dTMP molecules incorporated. Polymerase beta incorporates ddCMP with efficiency nearly equal to that of dCMP. HIV reverse transcriptase prefers to incorporate AZT-MP and ddCMP rather than dTMP and dCMP, respectively. The findings described here raise the concern that the capability of the two major host DNA polymerases to incorporate AZT-MP or ddCMP into DNA might cause adverse side effects on human DNA metabolism and mutation in the genomes of patients under long term continuous treatment with AZT and ddC.

Polymerases:

Topics:

Biotech Applications, Kinetic Parameters, Nucleotide Analogs / Template Lesions, Nucleotide Incorporation, Source / Purification

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Human Pol beta Copeland WC1992 Incorporation of non-standard nucleotides 95% + Nucleotide analog: 2',3'-Dideoxyribonucleotide nucleoside triphosphate
Human Pol beta Copeland WC1992 Cloned or native Native organism
Human Pol beta Copeland WC1992 Tagged Unspecified
Human Pol beta Copeland WC1992 Full length or truncated Unspecified
Human Pol beta Copeland WC1992 KM 2.6uM Reaction: Nucleotide incorporation; Substrate: dTTP; Technique: Steady State (Standing start primer template)
Human Pol beta Copeland WC1992 KM 1.7uM Reaction: Nucleotide incorporation; Substrate: dCTP; Technique: Steady State (Standing start primer template)
Human Pol beta Copeland WC1992 KM 1.2uM Reaction: Nucleotide incorporation; Substrate: ddCTP; Technique: Steady State (Standing start primer template)
Human Pol beta Copeland WC1992 KM 2.3uM Reaction: Nucleotide incorporation; Substrate: dTTP; Technique: Steady State (Activated DNA)
Human Pol beta Copeland WC1992 KM 1.5uM Reaction: Nucleotide incorporation; Substrate: dCTP; Technique: Steady State (Activated DNA)
Human Pol beta Copeland WC1992 Vmax 1.5 /second Reaction: Nucleotide incorporation; Substrate: dTTP; Technique: Steady State (Standing start primer template)
Human Pol beta Copeland WC1992 Vmax 27 /second Reaction: Nucleotide incorporation; Substrate: dCTP; Technique: Steady State (Standing start primer template)
Human Pol beta Copeland WC1992 Vmax 18 /second Reaction: Nucleotide incorporation; Substrate: ddCTP; Technique: Steady State (Standing start primer template)
Human Pol beta Copeland WC1992 Application name PCR
Human Pol beta Copeland WC1992 Kd 810uM Reaction: Nucleotide incorporation; Substrate: TTP analog; Technique: Steady State (Activated DNA)
Human Pol beta Copeland WC1992 Kd 2uM Reaction: Nucleotide incorporation; Substrate: ddCTP; Technique: Steady State (Activated DNA)
Human Pol alpha Copeland WC1992 Incorporation of non-standard nucleotides < 10% Nucleotide analog: Acycloriboucleoside-5'-O-(1-Thiotriphosphate)
Human Pol alpha Copeland WC1992 Incorporation of non-standard nucleotides Unspecified Nucleotide analog: 2',3'-Dideoxyribonucleotide nucleoside triphosphate
Human Pol alpha Copeland WC1992 Cloned or native Native organism
Human Pol alpha Copeland WC1992 Tagged Unspecified
Human Pol alpha Copeland WC1992 Full length or truncated Unspecified
Human Pol alpha Copeland WC1992 KM 1.5uM Reaction: Nucleotide incorporation; Substrate: dTTP; Technique: Steady State (Activated DNA)
Human Pol alpha Copeland WC1992 KM 1.8uM Reaction: Nucleotide incorporation; Substrate: dCTP; Technique: Steady State (activated DNA)
Human Pol alpha Copeland WC1992 KM 110uM Reaction: Nucleotide incorporation; Substrate: TTP analog; Technique: Steady State (Running start primer template)
Human Pol alpha Copeland WC1992 KM 1.4uM Reaction: Nucleotide incorporation; Substrate: dTTP; Technique: Steady State (Running start primer template)
Human Pol alpha Copeland WC1992 KM 2.1uM Reaction: Nucleotide incorporation; Substrate: dCTP; Technique: Steady State (Running start primer template)
Human Pol alpha Copeland WC1992 Vmax 2 /second Reaction: Nucleotide incorporation; Substrate: dTTP; Technique: Steady State (Running start primer template)
Human Pol alpha Copeland WC1992 Vmax 0.06 /second Reaction: Nucleotide incorporation; Substrate: TTP analog; Technique: Steady State (Running start primer template)
Human Pol alpha Copeland WC1992 Vmax 1.7 /second Reaction: Nucleotide incorporation; Substrate: dCTP; Technique: Steady State (Running start primer template)
Human Pol alpha Copeland WC1992 Application name PCR
Human Pol alpha Copeland WC1992 Kd 120uM Reaction: Nucleotide incorporation; Substrate: ddCTP; Technique: Steady State (Activated DNA)
Human Pol alpha Copeland WC1992 Kd 650uM Reaction: Nucleotide incorporation; Substrate: TTP analog; Technique: Steady State (Activated DNA)

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