Trace amounts of 8-oxo-dGTP in mitochondrial dNTP pools reduce DNA polymerase γ replication fidelity

Replication of the mitochondrial genome by DNA polymerase γ requires dNTP precursors that are subject to oxidation by reactive oxygen species generated by the mitochondrial respiratory chain. One such oxidation product is 8-oxo-dGTP, which can compete with dTTP for incorporation opposite template adenine to yield A-T to C-G transversions. Recent reports indicate that the ratio of undamaged dGTP to dTTP in mitochondrial dNTP pools from rodent tissues varies from ∼1:1 to >100:1. Within this wide range, we report here the proportion of 8-oxo-dGTP in the dNTP pool that would be needed to reduce the replication fidelity of human DNA polymerase γ. When various in vivo mitochondrial dNTP pools reported previously were used here in reactions performed in vitro, 8-oxo-dGTP was readily incorporated opposite template A and the resulting 8-oxo-G-A mismatch was not proofread efficiently by the intrinsic 3′ exonuclease activity of pol γ. At the dNTP ratios reported in rodent tissues, whether highly imbalanced or relatively balanced, the amount of 8-oxo-dGTP needed to reduce fidelity was <1% of dGTP. Moreover, direct measurements reveal that 8-oxo-dGTP is present at such concentrations in the mitochondrial dNTP pools of several rat tissues. The results suggest that oxidized dNTP precursors may contribute to mitochondrial mutagenesis in vivo, which could contribute to mitochondrial dysfunction and disease

Regulation of B family DNA polymerase fidelity by a conserved active site residue: characterization of M644W, M644L and M644F mutants of yeast DNA polymerase ε

To better understand the functions and fidelity of DNA polymerase ε (Pol ε), we report here on the fidelity of yeast Pol ε mutants with leucine, tryptophan or phenylalanine replacing Met644. The Met644 side chain interacts with an invariant tyrosine that contacts the sugar of the incoming dNTP. M644W and M644L Pol ε synthesize DNA with high fidelity, but M644F Pol ε has reduced fidelity resulting from strongly increased misinsertion rates. When Msh6-dependent repair of replication errors is defective, the mutation rate of a pol2-M644F strain is 16-fold higher than that of a strain with wild-type Pol ε. In conjunction with earlier studies of low-fidelity mutants with replacements for the homologous amino acid in yeast Pol α (L868M/F) and Pol δ (L612M), these data indicate that the active site location occupied by Met644 in Pol ε is a key determinant of replication fidelity by all three B family replicative polymerases. Interestingly, error specificity of M644F Pol ε is distinct from that of L868M/F Pol α or L612M Pol δ, implying that each polymerase has different active site geometry, and suggesting that these polymerase alleles may generate distinctive mutational signatures for probing functions in vivo

Light-strand bias and enriched zones of embedded ribonucleotides are associated with DNA replication and transcription in the human-mitochondrial genome

Abundant ribonucleoside-triphosphate (rNTP) incorporation into DNA by DNA polymerases in the form of ribonucleoside monophosphates (rNMPs) is a widespread phenomenon in nature, resulting in DNA-structural change and genome instability. The rNMP distribution, characteristics, hotspots and association with DNA metabolic processes in human mitochondrial DNA (hmtDNA) remain mostly unknown. Here, we utilize the ribose-seq technique to capture embedded rNMPs in hmtDNA of six different cell types. In most cell types, the rNMPs are preferentially embedded on the light strand of hmtDNA with a strong bias towards rCMPs; while in the liver-tissue cells, the rNMPs are predominately found on the heavy strand. We uncover common rNMP hotspots and conserved rNMP-enriched zones across the entire hmtDNA, including in the control region, which links the rNMP presence to the frequent hmtDNA replication-failure events. We show a strong correlation between coding-sequence size and rNMP-embedment frequency per nucleotide on the non-template, light strand in all cell types, supporting the presence of transient RNA-DNA hybrids preceding light-strand replication. Moreover, we detect rNMP-embedment patterns that are only partly conserved across the different cell types and are distinct from those found in yeast mtDNA. The study opens new research directions to understand the biology of hmtDNA and genomic rNMPs.Graphical Abstrac

Mismatch Repair–Independent Increase in Spontaneous Mutagenesis in Yeast Lacking Non-Essential Subunits of DNA Polymerase ε

Yeast DNA polymerase ε (Pol ε) is a highly accurate and processive enzyme that participates in nuclear DNA replication of the leading strand template. In addition to a large subunit (Pol2) harboring the polymerase and proofreading exonuclease active sites, Pol ε also has one essential subunit (Dpb2) and two smaller, non-essential subunits (Dpb3 and Dpb4) whose functions are not fully understood. To probe the functions of Dpb3 and Dpb4, here we investigate the consequences of their absence on the biochemical properties of Pol ε in vitro and on genome stability in vivo. The fidelity of DNA synthesis in vitro by purified Pol2/Dpb2, i.e. lacking Dpb3 and Dpb4, is comparable to the four-subunit Pol ε holoenzyme. Nonetheless, deletion of DPB3 and DPB4 elevates spontaneous frameshift and base substitution rates in vivo, to the same extent as the loss of Pol ε proofreading activity in a pol2-4 strain. In contrast to pol2-4, however, the dpb3Δdpb4Δ does not lead to a synergistic increase of mutation rates with defects in DNA mismatch repair. The increased mutation rate in dpb3Δdpb4Δ strains is partly dependent on REV3, as well as the proofreading capacity of Pol δ. Finally, biochemical studies demonstrate that the absence of Dpb3 and Dpb4 destabilizes the interaction between Pol ε and the template DNA during processive DNA synthesis and during processive 3′ to 5′exonucleolytic degradation of DNA. Collectively, these data suggest a model wherein Dpb3 and Dpb4 do not directly influence replication fidelity per se, but rather contribute to normal replication fork progression. In their absence, a defective replisome may more frequently leave gaps on the leading strand that are eventually filled by Pol ζ or Pol δ, in a post-replication process that generates errors not corrected by the DNA mismatch repair system

Comparison of changes to a conserved motif A residue and the contributions to fidelity of B family replicative DNA polymerases

<p><b>Copyright information:</b></p><p>Taken from "Regulation of B family DNA polymerase fidelity by a conserved active site residue: characterization of M644W, M644L and M644F mutants of yeast DNA polymerase "</p><p></p><p>Nucleic Acids Research 2007;35(9):3076-3086.</p><p>Published online 22 Apr 2007</p><p>PMCID:PMC1888828.</p><p>© 2007 The Author(s)</p> () Error rates are shown for exonuclease-proficient M644F Pol ε (; this study) and L612M Pol δ [; ()]. Values are for T·dTMP, A·dAMP, T·dGMP and G·dTMP base–base mismatches, as well as single-base deletions (−1). () Error rates are shown for exonuclease-deficient M644F Pol ε [; this study) and L868F Pol α (; ()]. Values are for T·dTMP, A·dAMP, T·dGMP and G·dTMP base–base mismatches, as well as single-base deletions (−1). The (≤) symbol indicates the error rate (or ratio) is less than or equal to the calculated value. values are shown above each comparison that was found to be statistically significant. The (* and ) symbols indicate values where  ≤ 0.01 and 0.001, respectively

Base substitution, −1, and +1 frameshift error rates for wild-type and M644F Pol ε

<p><b>Copyright information:</b></p><p>Taken from "Regulation of B family DNA polymerase fidelity by a conserved active site residue: characterization of M644W, M644L and M644F mutants of yeast DNA polymerase "</p><p></p><p>Nucleic Acids Research 2007;35(9):3076-3086.</p><p>Published online 22 Apr 2007</p><p>PMCID:PMC1888828.</p><p>© 2007 The Author(s)</p> Error rates for base substitution (B.S.), single-base deletions (−1) and single-base additions (+1) for exonuclease-proficient (left) and exonuclease-deficient Pol ε were calculated as described (). Error rates are shown for both wild-type Pol ε () and M644F Pol ε ()

Spectrum of single-base substitution and frameshift mutations made by M644F Pol ε in the gene

<p><b>Copyright information:</b></p><p>Taken from "Regulation of B family DNA polymerase fidelity by a conserved active site residue: characterization of M644W, M644L and M644F mutants of yeast DNA polymerase "</p><p></p><p>Nucleic Acids Research 2007;35(9):3076-3086.</p><p>Published online 22 Apr 2007</p><p>PMCID:PMC1888828.</p><p>© 2007 The Author(s)</p> Errors made by the exonuclease-proficient M644F Pol ε are shown above the sequence, while errors made by the exonuclease-deficient M644F Pol ε are shown below the sequence. Deletions are denoted by an and insertions by a . The numbering is in reference to the transcriptional start site (1)