Abstract
We have characterized, by transient-state kinetic methods, the polymerase and exonuclease activities of the human mitochondrial DNA polymerase (pol gamma) 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 (Kd) for nucleotide binding was increased 4-fold (12+/-1 microm). In addition, discrimination against mismatches was reduced approximately 20-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 (5.5x10(-5)+/-0.4x10(-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.
Highlights
31982 JOURNAL OF BIOLOGICAL CHEMISTRY mitochondrial genome [5], but the origin and significance of these ribonucleotides are unknown
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
The rate of the slow phase (Fig. 2A) is most probably a function of the release of the oligonucleotide duplex from the polymerase and subsequent rebinding of the enzyme to the substrate from solution. This weakened binding of the enzyme to the oligonucleotide duplex explains the low amplitudes of single nucleotide incorporation observed in these reactions
Summary
31982 JOURNAL OF BIOLOGICAL CHEMISTRY 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. Presteady state kinetic approaches have been used to begin to characterize more accurately the reverse transcriptase activity of human pol ␥ [6]. We demonstrate a marked reduction in fidelity during reverse transcription and show that the polymerase stalls after a single incorporation event
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