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.     

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.     

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.     

Reduction of dNTP levels enhances DNA replication fidelity in vivo

ATP is the most important energy source for the maintenance and growth of living cells. Here we report that the impairment of the aerobic respiratory chain by inactivation of the ndh gene, or the inhibition of glycolysis with arsenate, both of which reduce intracellular ATP, result in a significant decrease in spontaneous mutagenesis in Escherichia coli. The genetic analyses and mutation spectra in the ndh strain revealed that the decrease in spontaneous mutagenesis resulted from an enhanced accuracy of the replicative DNA polymerase. Quantification of the dNTP content in the ndh mutant cells and in the arsenate-treated cells showed reduction of the dNTP pool, which could explain the observed broad antimutator effects. In conclusion, our work indicates that the cellular energy supply could affect spontaneous mutation rates and that a reduction of the dNTP levels can be antimutagenic.     

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.     

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.     

On the fidelity of DNA replication. Lack of primer position effect on the fidelity of mammalian DNA polymerases

Mechanisms for the fidelity of DNA replication in eucaryotes are not adequately understood. Certain hypotheses can be tested by examining whether the first nucleotide inserted is incorporated with a significantly higher error rate than subsequent nucleotides. Using synthetic oligodeoxynucleotides, we have measured the effect of primer position on single-base misinsertion frequencies at an amber site in phi X174 DNA. Our results show a lack of position effect, indicating that processivity and the most direct "energy relay" proofreading mechanisms are not important determinants in eucaryotic 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.     

Lesion (in)tolerance reveals insights into DNA replication fidelity

The initial encounter of an unrepaired DNA lesion is likely to be with a replicative DNA polymerase, and the outcome of this event determines whether an error-prone or error-free damage avoidance pathway is taken. To understand the atomic details of this critical encounter, we have determined the crystal structures of the pol alpha family RB69 DNA polymerase with DNA containing the two most prevalent, spontaneously generated premutagenic lesions, an abasic site and 2'-deoxy-7,8-dihydro-8-oxoguanosine (8-oxodG). Identification of the interactions between these damaged nucleotides and the active site provides insight into the capacity of the polymerase to incorporate a base opposite the lesion. A novel open, catalytically inactive conformation of the DNA polymerase has been identified in the complex with a primed abasic site template. This structure provides the first molecular characterization of the DNA synthesis barrier caused by an abasic site and suggests a general mechanism for polymerase fidelity. In contrast, the structure of the ternary 8-oxodG:dCTP complex is almost identical to the replicating complex containing unmodified DNA, explaining the relative ease and fidelity by which this lesion is bypassed.     

DNA replication fidelity in Escherichia coli: a multi-DNA polymerase affair

High accuracy (fidelity) of DNA replication is important for cells to preserve the genetic identity and to prevent the accumulation of deleterious mutations. The error rate during DNA replication is as low as 10(-9) to 10(-11) errors per base pair. How this low level is achieved is an issue of major interest. This review is concerned with the mechanisms underlying the fidelity of the chromosomal replication in the model system Escherichia coli by DNA polymerase III holoenzyme, with further emphasis on participation of the other, accessory DNA polymerases, of which E.?coli contains four (Pols I, II, IV, and V). Detailed genetic analysis of mutation rates revealed that (1) Pol II has an important role as a back-up proofreader for Pol III, (2) Pols IV and V do not normally contribute significantly to replication fidelity, but can readily do so under conditions of elevated expression, (3) participation of Pols IV and V, in contrast to that of Pol II, is specific to the lagging strand, and (4) Pol I also makes a lagging-strand-specific fidelity contribution, limited, however, to the faithful filling of the Okazaki fragment gaps. The fidelity role of the Pol III τ subunit is also reviewed.     

Biological asymmetries and the fidelity of eukaryotic DNA replication.

A diploid human genome contains approximately six billion nucleotides. This enormous amount of genetic information can be replicated with great accuracy in only a few hours. However, because DNA strands are oriented antiparallel while DNA polymerization only occurs in the 5'----3' direction, semi-conservative replication of double-stranded DNA is an asymmetric process, i.e., there is a leading and a lagging strand. This provides a considerable opportunity for non-random error rates, because the architecture of the two strands as well as the DNA polymerases that replicate them may be different. In addition, the proteins that start or finish chains may well be different from those that perform the bulk of chain elongation. Furthermore, while replication fidelity depends on the absolute and relative concentrations of the four deoxyribonucleotide precursors, these are not equal in vivo, not constant throughout the cell cycle, and not necessarily equivalent in all cell types. Finally, the fidelity of DNA synthesis is sequence-dependent and the eukaryotic nuclear genome is a heterogeneous substrate. It contains repetitive and non-repetitive sequences and can actually be considered as two subgenomes that differ in nucleotide composition and gene content and that replicate at different times. The effects that each of these asymmetries may have on error rates during replication of the eukaryotic genome are discussed.     

Computational prediction of residues involved in fidelity checking for DNA synthesis in DNA polymerase I

Recent single-molecule F?rster resonance energy transfer studies of DNA polymerase I have led to the proposal of a postinsertion fidelity-checking site. This site is hypothesized to ensure proper base pairing of the newly inserted nucleotide. To help test this hypothesis, we have used energy decomposition, electrostatic free energy response, and noncovalent interaction analysis analyses to identify residues involved in this putative checking site. We have used structures of DNA polymerase I from two different organisms, the Klenow fragment from Escherichia coli and the Bacillus fragment from Bacillus stearothermophilus. Our results point to several residues that show altered interactions for three mispairs compared to the correctly paired DNA dimer. Furthermore, many of these residues are conserved among A family polymerases. The identified residues provide potential targets for mutagenesis studies for investigation of the fidelity-checking site hypothesis.     

Analysis of fidelity mechanisms with eukaryotic DNA replication and repair proteins

We are investigating the mechanisms for producing or avoiding errors during DNA synthesis catalyzed by DNA replication and repair proteins purified from eukaryotic sources. Using assays that monitor the fidelity of a single round of DNA synthesis in vitro, we have defined the error frequency and mutational specificity of the four classes of animal cell DNA polymerases (alpha, beta, delta, gamma), and the fidelity of an SV40 origin-dependent DNA replication complex in extracts of HeLa cells.     

Palm mutants in DNA polymerases alpha and eta alter DNA replication fidelity and translesion activity

We isolated active mutants in Saccharomyces cerevisiae DNA polymerase alpha that were associated with a defect in error discrimination. Among them, L868F DNA polymerase alpha has a spontaneous error frequency of 3 in 100 nucleotides and 570-fold lower replication fidelity than wild-type (WT) polymerase alpha. In vivo, mutant DNA polymerases confer a mutator phenotype and are synergistic with msh2 or msh6, suggesting that DNA polymerase alpha-dependent replication errors are recognized and repaired by mismatch repair. In vitro, L868F DNA polymerase alpha catalyzes efficient bypass of a cis-syn cyclobutane pyrimidine dimer, extending the 3' T 26000-fold more efficiently than the WT. Phe34 is equivalent to residue Leu868 in translesion DNA polymerase eta, and the F34L mutant of S. cerevisiae DNA polymerase eta has reduced translesion DNA synthesis activity in vitro. These data suggest that high-fidelity DNA synthesis by DNA polymerase alpha is required for genomic stability in yeast. The data also suggest that the phenylalanine and leucine residues in translesion and replicative DNA polymerases, respectively, might have played a role in the functional evolution of these enzyme classes.     

On the fidelity of DNA replication. Studies with human placenta DNA polymerases.

The fidelity of DNA synthesis with purified DNA polymerase alpha and beta from human placenta has been studied. With poly[d(A-T)] as the template-primer and Mg2+ as the metal activator, DNA polymerase alpha incorporates 1 mol of dGMP for every 6,000 to 12,000 mol of complementary nucleotides polymerized. Under the same conditions, DNA polymerase beta is more accurate, the error rate being 1/20,000 to 1/60,000. This greater accuracy of DNA polymerase beta is observed with a variety of homopolymer templates. With both enzymes, substitution of Mg2+ with activating concentrations of Mn2+ or Co2+ enhances the frequency of misincorporation. At greater than activating concentrations of Mn2+ and Co2+, there is an inhibition of complementary nucleotide incorporation, further increasing the frequency of misincorporation. Nearest neighbor analysis of the products synthesized with both enzymes indicates that the noncomplementary nucleotides are incorporated predominantly as single base substitutions. The greater accuracy of DNA polymerase beta over DNA polymerase alpha should be considered in relationship to their possible roles in DNA replication and repair.     

Balancing eukaryotic replication asymmetry with replication fidelity

Coordinated replication of eukaryotic nuclear genomes is asymmetric, with copying of a leading strand template preceding discontinuous copying of the lagging strand template. Replication is catalyzed by DNA polymerases α, δ and ?, enzymes that are related yet differ in physical and biochemical properties, including fidelity. Recent studies suggest that Pol ? is normally the primary leading strand replicase, whereas most synthesis by Pol δ occurs during lagging strand replication. New studies show that replication asymmetry can generate strand-specific genome instability resulting from biased deoxynucleotide pools and unrepaired ribonucleotides incorporated into DNA during replication, and that the eukaryotic replication machinery has evolved to most efficiently correct those replication errors that are made at the highest rates.