Fidelity and Processivity of Reverse Transcription by the Human Mitochondrial DNA Polymerase

Abstract

We have characterized, by transient-state kinetic methods, the polymerase and exonuclease activities of the human mitochondrial DNA polymerase (pol ␥) during reverse transcription, employing a synthetic oligonucleotide consisting of a DNA primer and an RNA template. In comparison with the kinetic parameters observed with a DNA template, the rate of correct deoxynucleotide incorporation was reduced 25-fold (5.5 ؎ 0.2 s ؊1 ), whereas the dissociation constant (K d ) for nucleotide binding was increased 4-fold (12 ؎ 1 M). In addition, discrimination against mismatches was reduced ϳ20-fold to only 15,000 on average. The proofreading exonuclease favored the removal of an incorrect nucleotide (0.0021 ؎ 0.0002 s ؊1 for correct versus 0.034 ؎ 0.004 s ؊1 for incorrect), and the partitioning between incorporation beyond a mismatch (5.5 ؋ 10 ؊5 ؎ 0.4 ؋ 10 ؊5 s ؊1 ), and exonuclease removal of that mismatch favors removal of the mismatch. These data suggest that the "reverse transcriptase activity" of mitochondrial polymerase could be physiologically relevant. However, the enzyme stalls and is unable to efficiently incorporate beyond a single nucleotide with an RNA template. Additionally, we present a refined method for calculating net discrimination, which more accurately describes the contributions of correct and incorrect incorporation. The biological and biotechnological significance of these results are discussed.Reverse transcription is the process of DNA polymerization using an RNA template, and it is interesting to note that mitochondrial DNA polymerases (pol ␥) were initially characterized by their ability to reverse-transcribe DNA from RNA (1). In fact, over the past 30 years, human pol 2 ␥ has been shown to be an effective reverse transcriptase in steady state single nucleotide incorporation assays using DNA/RNA heteroduplexes to probe for its presence and to assay its replication fidelity (1-4). RNase digestion of mammalian mitochondrial DNA has shown that there are at least 30 ribonucleotides incorporated in the mitochondrial genome (5), but the origin and significance of these ribonucleotides are unknown. They could be the products of incorporation by the DNA polymerase or merely remnants of RNA priming by the mitochondrial RNA polymerase after lagging strand synthesis and incomplete primer removal.Steady state assays have been used to characterize reverse transcriptase activity and fidelity of pol ␥ largely because steady state rates are faster with an RNA template. However, steady state, single nucleotide turnover rates are limited by polymerase dissociation from product complexes and do not provide reliable measures of nucleotide discrimination. Therefore, presteady state kinetic approaches have been used to begin to characterize more accurately the reverse transcriptase activity of human pol ␥ (6). In our previous studies we were surprised to see how rapidly pol ␥ catalyzed a single DNA incorporation with an RNA template. Here we demonstrate a marked reduction in fidelity during reverse transcription and s...

The interface of transcription and DNA replication in the mitochondria

“…Studies investigating the physiological consequence of reverse transcriptase activity by human pol γ opposite a single ribonucleotide found that pol γ stalls after incorporating a single deoxyribonucleotide [57]. In addition, recent results reveal that the efficiency of bypassing a single ribonucleotide in the template DNA was 51% compared to a control template containing no ribonucleotides [40].…”

Ribonucleotide Discrimination and Reverse Transcription by the Human Mitochondrial DNA Polymerase

“…Because it was previously shown that the affinity of the human DNA pol ␥ with RNA/DNA substrates is similar to that with DNA/DNA substrates, comparison of steady state kinetic parameters becomes valid (22,23). Reaction mixtures (10 l) contained 25 mM HEPES-KOH (pH 7.6), 2 mM 2-mercaptoethanol, 0.1 mM EDTA, 5 mM MgCl 2 , 50 nM radiolabeled substrate, 10 nM exonuclease-deficient WT or E895A Shown is a molecular model of the human DNA pol ␥ active site with incoming ddCTP (dark blue), and Mg 2ϩ (gray spheres).…”

“…Previous biochemical studies using purified DNA polymerase ␥ suggested that the enzyme is an efficient reverse transcriptase and possesses the ability to incorporate and extend ribonucleotides (22,23). However, a comprehensive analysis of incorporation, discrimination, extension, and bypass of ribonucleotides by DNA pol ␥ is still lacking.…”

A mechanistic view of human mitochondrial DNA polymerase γ: Providing insight into drug toxicity and mitochondrial disease

“…Furthermore, Pol γ can perform exonuclease cleavage of mismatched dNMPs opposite a RNA template [50]. The reverse transcriptase activity of Pol γ was later found to be not processive, incorporating one dNTP and dissociating from the primer/template [51]. This indicates that the physiological significance of this phenomenon may be linked to single ribonucelotides present throughout the template strand.…”

“…This indicates that the physiological significance of this phenomenon may be linked to single ribonucelotides present throughout the template strand. The weaker interaction between Pol γ and DNA/RNA primer-templates also serves to explain the increased steady-state rate of incorporation versus DNA/DNA primer-templates, as the steady-state rate is governed by the rate of primer-template release [51]. …”