Fidelity and processivity of reverse transcription by the human mitochondrial DNA polymerase.
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.
Lee
Harold R
HR
Department of Chemistry and Biochemistry, Institute of Cellular and Molecular Biology, University of Texas, Austin, Texas 78712, USA.
Johnson
Kenneth A
KA
D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism; D005092:Exonucleases / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D048348:Reverse Transcription
D003854:Deoxyribonucleotides; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; C413027:POLG protein, human; D005092:Exonucleases