Trace amounts of 8-oxo-dGTP in mitochondrial dNTP pools reduce DNA polymerase gamma 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.     

DNA replication fidelity

DNA replication fidelity plays fundamental role in faithful transmission of genetic material during cell division and during transfer of genetic material from parents to progeny. Replicative polymerases are the main guardian responsible for high replication fidelity of genomic DNA. DNA main replicative polymerases are also involved in many DNA repair processes. High fidelity of DNA replication is determined by correct nucleotide selectivity in polymerase active center, and exonucleolytic proofreading that removes mismatches from primer terminus. In this article we will focus on the mechanisms that are responsible for high fidelity of replications with the special emphasis on structural studies showing important conformational changes after substrate binding. We will also stress the importance of hydrogen bonding, base pair geometry, polymerase DNA interactions and the role of accessory proteins in replication fidelity.     

Role of Escherichia coli DNA polymerase IV in in vivo replication fidelity

We have investigated whether DNA polymerase IV (Pol IV; the dinB gene product) contributes to the error rate of chromosomal DNA replication in Escherichia coli. We compared mutation frequencies in mismatch repair-defective strains that were either dinB positive or dinB deficient, using a series of mutational markers, including lac targets in both orientations on the chromosome. Virtually no contribution of Pol IV to the chromosomal mutation rate was observed. On the other hand, a significant effect of dinB was observed for reversion of a lac allele when the lac gene resided on an F'(pro-lac) episome.     

Entropy involved in fidelity of DNA replication

Information has an entropic character which can be analyzed within the framework of the Statistical Theory in molecular systems. R. Landauer and C.H. Bennett showed that a logical copy can be carried out in the limit of no dissipation if the computation is performed sufficiently slowly. Structural and recent single-molecule assays have provided dynamic details of polymerase machinery with insight into information processing. Here, we introduce a rigorous characterization of Shannon Information in biomolecular systems and apply it to DNA replication in the limit of no dissipation. Specifically, we devise an equilibrium pathway in DNA replication to determine the entropy generated in copying the information from a DNA template in the absence of friction. Both the initial state, the free nucleotides randomly distributed in certain concentrations, and the final state, a polymerized strand, are mesoscopic equilibrium states for the nucleotide distribution. We use empirical stacking free energies to calculate the probabilities of incorporation of the nucleotides. The copied strand is, to first order of approximation, a state of independent and non-indentically distributed random variables for which the nucleotide that is incorporated by the polymerase at each step is dictated by the template strand, and to second order of approximation, a state of non-uniformly distributed random variables with nearest-neighbor interactions for which the recognition of secondary structure by the polymerase in the resultant double-stranded polymer determines the entropy of the replicated strand. Two incorporation mechanisms arise naturally and their biological meanings are explained. It is known that replication occurs far from equilibrium and therefore the Shannon entropy here derived represents an upper bound for replication to take place. Likewise, this entropy sets a universal lower bound for the copying fidelity in replication.     

DNA replication fidelity: proofreading in trans

Proofreading is the primary guardian of DNA polymerase fidelity. New work has revealed that polymerases with intrinsic proofreading activity may cooperate with non-proofreading polymerases to ensure faithful DNA replication.     

Prechemistry versus preorganization in DNA replication fidelity

The molecular origin of nucleotide insertion catalysis and fidelity of DNA polymerases is explored by means of computational simulations. Special attention is paid to the examination of the validity of proposals that invoke prechemistry effects, checkpoints concepts, and dynamical effects. The simulations reproduce the observed fidelity in Pol β, starting with the relevant observed X-ray structures of the complex with the right (R) and wrong (W) nucleotides. The generation of free energy surfaces for the R and W systems also allowed us to analyze different proposals about the origin of the fidelity and to reach several important conclusions. It is found that the potential of mean force (PMF) obtained by proper sampling does not support QM/MM-based proposals of a large barrier before the prechemistry state. Furthermore, examination of dynamical proposals by the renormalization approach indicates that the motions from open to close configurations do not contribute to catalysis or fidelity. Finally we discuss and analyze the induced fit concept and show that, despite its importance, it does not explain fidelity. That is, the fidelity is apparently due to the change in the preorganization of the chemical site, as a result of the relaxation of the binding site upon binding of the incorrect nucleotide. Finally and importantly, since the issue is the barrier associated with the enzyme-substrate (ES)/DNA complex at the chemical transition state and not the path to this complex formation (unless this path involves rate determining steps), it is also not useful to invoke checkpoints while discussing fidelity.     

DNA replication fidelity: kinetics and thermodynamics

Mechanisms that control the fidelity of DNA replication are discussed. Data are reviewed for 3 steps in a fidelity pathway: nucleotide insertion, exonucleolytic proofreading, and extension from matched and mismatched 3′-primer termini. Fidelity mechanisms that involve predominately K_m discrimination, V_max discrimination, or a combination of the two are analyzed in the context of a simple model for fidelity. Each fidelity step is divided into 2 components, thermodynamics and kinetic. The thermodynamic component, which relates to free-energy differences between right and wrong base pair, is associated with a K_m discrimination mechanism for polymerase. The kinetic component, which represents the enzyme's ability to select bases for insertion and excision to achieve fidelity greater than that availablek from base pairing free-energy differences, is associated with a V_max discrimination mechanism for polymerase. Currently available fidelity data for nucleotide insertion and primer extension in the absence of proofreading appears to have relatively large K_m and small V_max components. An important complication can arise when analyzing data from polymerases containing an associated 3′-exonuclease activity. In the presence of proofreading, a V_max discrimination mechanisms is likely to occur, but this may be the result of two K_m discrimination mechanisms acting serially, one for nucleotide insertion and other for excision. Possible relationships between base pairing free energy differences measured in aqueous solution and those defined within the polymerase active cleft are considered in the context of the enzyme's ability to exclude water, at least partially, from the vicinity of its active site.     

Role of Escherichia coli DNA polymerase I in chromosomal DNA replication fidelity

We have investigated the possible role of Escherichia coli DNA polymerase (Pol) I in chromosomal replication fidelity. This was done by substituting the chromosomal polA gene by the polAexo variant containing an inactivated 3′→5′ exonuclease, which serves as a proofreader for this enzyme's misinsertion errors. Using this strain, activities of Pol I during DNA replication might be detectable as increases in the bacterial mutation rate. Using a series of defined lacZ reversion alleles in two orientations on the chromosome as markers for mutagenesis, 1.5‐ to 4‐fold increases in mutant frequencies were observed. In general, these increases were largest for lac orientations favouring events during lagging strand DNA replication. Further analysis of these effects in strains affected in other E. coli DNA replication functions indicated that this polAexo mutator effect is best explained by an effect that is additive compared with other error‐producing events at the replication fork. No evidence was found that Pol I participates in the polymerase switching between Pol II, III and IV at the fork. Instead, our data suggest that the additional errors produced by polAexo are created during the maturation of Okazaki fragments in the lagging strand.     

Hydroxyurea arrests DNA replication by a mechanism that preserves basal dNTP pools

The relationship between dNTP levels and DNA synthesis was investigated using alpha factor-synchronized yeast treated with the ribonucleotide reductase inhibitor hydroxyurea (HU). Although HU blocked DNA synthesis and prevented the dNTP pool expansion that normally occurs at G1/S, it did not exhaust the levels of any of the four dNTPs, which dropped to about 80% of G1 levels. When dbf4 yeast that are ts for replication initiation were allowed to preaccumulate dNTPs at 37 degrees C before being released to 25 degrees C in the presence of HU, they synthesized 0.3 genome equivalents of DNA and then arrested as dNTPs approached sub-G1 levels. Accumulation of dNTPs at G1/S was not a prerequisite for replication initiation, since dbf4 cells incubated in HU at 25 degrees C were able to replicate when subsequently switched to 37 degrees C in the absence of HU. The replication arrest mechanism was not dependent on the Mec1/Rad53 pathway, since checkpoint-deficient rad53 cells also failed to exhaust basal dNTPs when incubated in HU. The persistence of basal dNTP levels in HU-arrested cells and partial bypass of the arrest in cells that had preaccumulated dNTPs suggest that cells have a mechanism for arresting DNA chain elongation when dNTP levels are not maintained above a critical threshold.     

DNA polymerase beta: contributions of template-positioning and dNTP triphosphate-binding residues to catalysis and fidelity

The specific catalytic roles of two groups of DNA polymerase beta active site residues identified from crystal structures were investigated: residues possibly involved in DNA template positioning (Lys280, Asn294, and Glu295) and residues possibly involved in binding the triphosphate moiety of the incoming dNTP (Arg149, Ser180, Arg183, and Ser188). Eight site-specific mutants were constructed: K280A, N294A, N294Q, E295A, R149A, S180A, R183A, and S188A. Two-dimensional NMR analysis was employed to show that the global conformation of the mutants has not been perturbed significantly. Pre-steady-state kinetic analyses with single-nucleotide gapped DNA substrates were then performed to obtain the rate of catalysis at saturating dNTP (k(pol)), the apparent dissociation constant for dNTP (K(d)), catalytic efficiency k(pol)/K(d), and fidelity. Of the three template-positioning residues, Asn294 and Glu295 (but not Lys280) contribute significantly to k(pol). Taken together with other data, the results suggest that these two residues help to stabilize the transition state during catalysis even though they interact with the DNA template backbone rather than directly with the incoming dNTP or the opposite base on the template. Furthermore, the fidelity increases by up to 19-fold for N294Q due to differential k(pol) effects between correct and incorrect nucleotides. Of the four potential triphosphate-binding residues, Ser180 and Arg183 contribute significantly to k(pol) while the effects of R149A are relatively small and are primarily on K(d), and Ser188 appears to play a minimal role in the catalysis by Pol beta. These results identify several residues important for catalysis and quantitate the contributions of each of those residues. The functional data are discussed in relation to the prediction on the basis of available crystal structures.     

On the fidelity of DNA replication. Isolation of high fidelity DNA polymerase-primase complexes by immunoaffinity chromatography

Error rates for conventionally purified DNA polymerase-alpha from calf thymus, chicken, and human sources have been reported to be one in 10,000 to one in 40,000 nucleotides incorporated. Isolation of polymerase-alpha by immunoaffinity chromatography yields a multiprotein high molecular weight replication complex that contains an associated DNA primase (Wong, S. W., Paborsky, L. R., Fisher, P. A., Wang, T. S-F., and Korn, D. (1986) J. Biol. Chem. 261, 7958-7968). We have isolated DNA polymerase-primase complexes from calf thymus, from a human lymphoblast cell line (TK-6), and from Chinese hamster lung cells (V-79) using two different methods of immunoaffinity chromatography. These enzyme complexes are 12- to 20-fold more accurate than conventionally purified calf thymus DNA polymerase-alpha when assayed using the phi X174am3 fidelity assay; estimated error rates are one in 460,000 to one in 830,000 nucleotides incorporated when the enzyme complex is freshly isolated. The polymerase-primase complex from calf thymus exhibited no detectable 3'----5' exonuclease activity using a heteroduplex substrate containing a single 3'-terminal mismatched nucleotide. Upon prolonged storage at -70 degrees C, the error rate of the immunoaffinity-purified calf thymus DNA polymerase-primase complex increases to about one in 50,000 nucleotides incorporated, an error rate similar to that exhibited by conventional isolates of DNA polymerase-alpha.     

Endogenous DNA replication stress results in expansion of dNTP pools and a mutator phenotype

The integrity of the genome depends on diverse pathways that regulate DNA metabolism. Defects in these pathways result in genome instability, a hallmark of cancer. Deletion of ELG1 in budding yeast, when combined with hypomorphic alleles of PCNA results in spontaneous DNA damage during S phase that elicits upregulation of ribonucleotide reductase (RNR) activity. Increased RNR activity leads to a dramatic expansion of deoxyribonucleotide (dNTP) pools in G1 that allows cells to synthesize significant fractions of the genome in the presence of hydroxyurea in the subsequent S phase. Consistent with the recognized correlation between dNTP levels and spontaneous mutation, compromising ELG1 and PCNA results in a significant increase in mutation rates. Deletion of distinct genome stability genes RAD54, RAD55, and TSA1 also results in increased dNTP levels and mutagenesis, suggesting that this is a general phenomenon. Together, our data point to a vicious circle in which mutations in gatekeeper genes give rise to genomic instability during S phase, inducing expansion of the dNTP pool, which in turn results in high levels of spontaneous mutagenesis.     

Central carbon metabolism influences fidelity of DNA replication in Escherichia coli

Recent studies indicated that there is a direct link between central carbon metabolism (CCM) and initiation and elongation of DNA replication in Eschericha coli. Namely, effects of certain mutations in genes coding for replication proteins (dnaA, dnaB, dnaE, dnaG, and dnaN) could be specifically suppressed by deletions of some genes, whose products are involved in CCM reactions (pta, ackA, pgi, tktB, and gpmA). Here, we demonstrate that the link between CCM and DNA synthesis can be extended to the DNA replication fidelity, as we report changes of the mutator phenotypes of E. coli dnaQ49 and dnaX36 mutants (either suppression or enhancement) by dysfunctions of zwf, pta, ackA, acnB, and icdA genes. Overexpression of appropriate wild-type CCM genes in double mutants resulted in reversions to the initial mutator phenotypes, indicating that the effects were specific. Moreover, the observed suppression and enhancement effects were not caused by changes in bacterial growth rates. These results suggest that there is a genetic correlation between CCM and DNA replication fidelity in E. coli, apart from the already documented link between CCM and DNA replication initiation control and elongation efficiency.     

On the fidelity of DNA replication: manganese mutagenesis in vitro

Manganese is mutagenic in vivo and in vitro in studies with a variety of enzymes and templates. Using Escherichia coli DNA polymerase I with poly[d(A-T)] and phi X174 DNA templates, we analyzed the mechanism of manganese mutagenesis by determining the dependence of error rate on free Mn2+ concentration and comparing this to measured dissociation constants of Mn2+ from enzyme, template, and deoxynucleoside triphosphate substrates. This comparison suggests several conclusions: (1) At very low Mn2+ concentrations, the enzyme is activated at high fidelity. Thus, it is unlikely that activation with manganese per se significantly alters the conformation of the enzyme so as to affect nucleotide selection. (2) At low free Mn2+ concentrations (less than 100 microM), manganese causes errors in incorporation via its interaction with the DNA template. The concentration dependence of mutagenesis is determined by the strength of binding Mn2+ to the particular DNA template used. The data do not allow one to rule out the possibility that Mn2+-deoxynucleoside triphosphate interactions contribute to mutagenesis in selected situations. This range of free Mn2+ concentrations is the one of greatest relevance for in vivo mutagenesis. (3) At higher concentrations (between 500 microM and 1.5 mM), further mutagenesis by Mn2+ occurs. This mutagenesis probably is due either to binding of manganese to single-stranded regions within the DNA or to weak accessory sites on the enzyme.