Abstract
During DNA synthesis, DNA polymerases must select against ribonucleotides, present at much higher levels compared with deoxyribonucleotides. Most DNA polymerases are equipped to exclude ribonucleotides from their active site through a bulky side chain residue that can sterically block the 2'-hydroxyl group of the ribose ring. However, many nuclear replicative and repair DNA polymerases incorporate ribonucleotides into DNA, suggesting that the exclusion mechanism is not perfect. In this study, we show that the human mitochondrial DNA polymerase γ discriminates ribonucleotides efficiently but differentially based on the base identity. Whereas UTP is discriminated by 77,000-fold compared with dTTP, the discrimination drops to 1,100-fold for GTP versus dGTP. In addition, the efficiency of the enzyme was reduced 3-14-fold, depending on the identity of the incoming nucleotide, when it extended from a primer containing a 3'-terminal ribonucleotide. DNA polymerase γ is also proficient in performing single-nucleotide reverse transcription reactions from both DNA and RNA primer terminus, although its bypass efficiency is significantly diminished with increasing stretches of ribonucleotides in template DNA. Furthermore, we show that the E895A mutant enzyme is compromised in its ability to discriminate ribonucleotides, mainly due to its defects in deoxyribonucleoside triphosphate binding, and is also a poor reverse transcriptase. The potential biochemical defects of a patient harboring a disease mutation in the same amino acid (E895G) are discussed.
Highlights
Replicative DNA polymerases must have the ability to discriminate between the correct and incorrect nucleotide to be incorporated during DNA synthesis and to distinguish between the correct and incorrect sugar moiety to maintain its fidelity
We show that the human mitochondrial DNA polymerase ␥ discriminates ribonucleotides efficiently but differentially based on the base identity
Ribonucleotides Are Strongly and Differentially Discriminated by Human DNA Pol ␥—At least 30 ribonucleotides are found scattered in the mitochondrial genome [27], and it has been shown that DNA pol ␥ can incorporate a single ribonucleotide into a DNA primer [22], suggesting a role for pol ␥ in this activity
Summary
Materials—Ultrapure dNTP and NTP solutions were obtained from Amersham Biosciences, and [␥-32P]ATP was from PerkinElmer Life Sciences. Expression and Purification—The His affinity-tagged recombinant catalytic subunits of WT and E895A human pol ␥ were overproduced in baculovirus-infected Sf9 cells, and the proteins were purified to homogeneity as described previously [20, 24, 25]. Kinetic Assays—Steady state kinetic parameters were determined using a polyacrylamide gel-based single-nucleotide extension assay. 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]. Ribonucleotide Bypass Assays—Bypass reactions were performed similar to the single-nucleotide extension assays with the following exceptions: (i) all four dNTPs were added to the reaction mixtures, and (ii) the incubation time was 5 min at 37 °C
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