Ribonucleotide Discrimination and Reverse Transcription by the Human Mitochondrial DNA Polymerase

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 ␥ 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 ␥ 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.Replicative DNA polymerases must have the ability not only to discriminate between the correct and incorrect nucleotide to be incorporated during DNA synthesis but also to distinguish between the correct and incorrect sugar moiety to maintain its fidelity. This process is crucial because ribonucleotides are more prone to strand cleavage compared with deoxyribonucleotides due to the presence of a reactive hydroxyl group at the 2Ј-position of the ribose sugar. The discrimination against ribonucleotide incorporation during various DNA metabolic processes has been studied for many nuclear replicative DNA polymerases, including pol 2 ␣, ␦, and ⑀ and other polymerases involved in DNA repair like pol ␤, , and (1-5). Studies on these eukaryotic nuclear replicative and repair enzymes indicate that the levels of ribonucleotide discrimination vary from 2 to 3 orders of magnitude between them, suggesting that the mechanism employed by each polymerase in excluding ribonucleotide from its active site may be different during DNA synthesis. Analysis of the human DNA polymerase ␤ revealed that this polymerase discriminates against ribonucleotide incorporation by nearly 4 orders of magnitude (3). DNA pol ␥, encoded by the nuclear gene POLG, is the sole DNA polymerase in human mitochondria and hence bears the burden of replicating and repairing the entire mitochondrial DNA (6, 7). The holoenzyme of pol ␥ is a heterotrimer composed of a catalytic subunit (p140) and a homodimeric accessory subunit (p55...

Structural Factors That Determine Selectivity of a High Fidelity DNA Polymerase for Deoxy-, Dideoxy-, and Ribonucleotides

“…These values are consistent with those determined for other DNA polymerases in the A family (14,19,50,51). Wild-type BF favors dCTP incorporation over rCTP or ddCTP by 4 and 3 orders of magnitude, respectively.…”

Primer retention owing to the absence of RNase H1 is catastrophic for mitochondrial DNA replication

“…The persistent RNAs are capable of being fully incorporated into mtDNA. However, although mitochondrial DNA polymerase [DNA polymerase γ (POLG)] copes well with a single ribonucleotide in a DNA template, more extensive RNA patches are a major impediment to its ability to synthesize DNA (29). Thus, retained RNA primers in mtDNA are expected to cause replication stalling and arrest and prevent the completion, and ultimately the initiation, of subsequent rounds of replication.…”

“…Primers on the H-strand from the LSP to the origin of replication Ori-H (or Ori-b, not illustrated) persist when RNase H1 is absent, and these are fully incorporated into mtDNA at the end of the replication cycle. In the next round of replication, the mitochondrial DNA polymerase γ (POLG) encounters a stretch of 150 (or 550) ribonucleotides of which it is expected to be able to synthesize only one or a few ribonucleotides, as it is a poor RNA-dependent DNA polymerase (29). RNase H1 ablation revealed a RNA patch on the L-strand near LSP that will prevent completion of H-strand synthesis once it is incorporated in the template strand.…”

The Balancing Act of Ribonucleotides in DNA

“…The abilities of several eukaryotic polymerases to incorporate, extend and bypass ribonucleotides in DNA are summarized in Table 1[2, 6, 8, 12, 14, 19, 20, 22, 26-33]. …”