High fidelity vertical transmission of genetic information: DNA replication and repair.
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DNA Repair↗
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Trading places: how do DNA polymerases switch during translesion DNA synthesis?
The replicative bypass of base damage in DNA (translesion DNA synthesis [TLS]) is a ubiquitous mechanism for relieving arrested DNA replication. The process requires multiple polymerase switching events during which the high-fidelity DNA polymerase in the replication machinery arrested at the primer terminus is replaced by one or more polymerases that are specialized for TLS. When replicative bypass is fully completed, the primer terminus is once again occupied by high-fidelity polymerases in the replicative machinery. This review addresses recent advances in our understanding of DNA polymerase switching during TLS in bacteria such as E. coli and in lower and higher eukaryotes.
Bacterial Proteins↗
African swine fever virus dUTPase is a highly specific enzyme required for efficient replication in swine macrophages.
The African swine fever virus (ASFV) gene E165R, which is homologous to dUTPases, has been characterized. A multiple alignment of dUTPases showed the conservation in ASFV dUTPase of the motifs that define this protein family. A biochemical analysis of the purified recombinant enzyme showed that the virus dUTPase is a trimeric, highly specific enzyme that requires a divalent cation for activity. The enzyme is most probably complexed with Mg(2+), the preferred cation, and has an apparent K(m) for dUTP of 1 microM. Northern and Western blotting, as well as immunofluorescence analyses, indicated that the enzyme is expressed at early and late times of infection and is localized in the cytoplasm of the infected cells. On the other hand, an ASFV dUTPase-deletion mutant (vDeltaE165R) has been obtained. Growth kinetics showed that vDeltaE165R replicates as efficiently as parental virus in Vero cells but only to 10% or less of parental virus in swine macrophages. Our results suggest that the dUTPase activity is dispensable for virus replication in dividing cells but is required for productive infection in nondividing swine macrophages, the natural host cell for the virus. The viral dUTPase may play a role in lowering the dUTP concentration in natural infections to minimize misincorporation of deoxyuridine into the viral DNA and ensure the fidelity of genome replication.
African Swine Fever↗
Structural basis for the dual coding potential of 8-oxoguanosine by a high-fidelity DNA polymerase.
Accurate DNA replication involves polymerases with high nucleotide selectivity and proofreading activity. We show here why both fidelity mechanisms fail when normally accurate T7 DNA polymerase bypasses the common oxidative lesion 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8oG). The crystal structure of the polymerase with 8oG templating dC insertion shows that the O8 oxygen is tolerated by strong kinking of the DNA template. A model of a corresponding structure with dATP predicts steric and electrostatic clashes that would reduce but not eliminate insertion of dA. The structure of a postinsertional complex shows 8oG(syn).dA (anti) in a Hoogsteen-like base pair at the 3' terminus, and polymerase interactions with the minor groove surface of the mismatch that mimic those with undamaged, matched base pairs. This explains why translesion synthesis is permitted without proofreading of an 8oG.dA mismatch, thus providing insight into the high mutagenic potential of 8oG.
Bacteriophage T7↗
Separate sexes and the mitochondrial theory of ageing.
An hypothesis is presented by which gamete specialization resolves a conflict between the function and replication of mitochondria. The function of mitochondria is to synthesize ATP by oxidative phosphorylation, which is coupled to respiratory electron transport. This requires a mitochondrial genetic system. However, "incorrect" electron transfers produce free radicals that cause mutation, and the frequency of these events is increased by mutation. Mitochondrial function is therefore detrimental to the fidelity of mitochondrial replication. Damage to somatic mitochondrial DNA may accumulate within, and indeed determine, the life span of individual organisms. Motility of one gamete is required for fertilization, and requires ATP. It is proposed that male gametes maximize energy production for motility by sacrificing mitochondrial DNA to electron transfer and its mutagenic by-products, while female gametes, which are non-motile, repress mitochondrial oxidative phosphorylation, thus protecting mitochondrial DNA for faithful transmission between generations. Male gametes then make no contribution to the mitochondrial genome of the zygote: mitochondria are maternally inherited. This testable hypothesis may help to explain the evolution of separate sexes and a number of their characteristics. Maternal inheritance of chloroplasts may be explained in a similar way, and contribute to the maintenance of separate sexes in plants.
Aging↗
Infidelity of DNA synthesis as a cause of mutagenesis.
The concept underlying these studies is that a major determinant of mutagenesis involves perturbations in the fidelity of DNA replication. i.e., the accuracy by which DNA polymerases copy DNA templates. To investigate this relationship, we have designed in vitro assays to measure the accuracy of DNA replication and used these systems to screen for and to quantitate factors that promote errors in DNA synthesis. Using DNA polymerase from bacteria, the frequency of mistakes with phi X174 DNA as a template approaches 10(-7) and is similar to the spontaneous mutation rates in bacterial cells. In contrast, DNA polymerases from animal cells are more error-prone. The differences in fidelity among mammalian DNA polymerases which lack error-correcting mechanisms suggest that these enzymes enhance accuracy by improving base-selection. Thus, mutants in DNA polymerase-alpha might be altered in base-selection. Chinese hamster V79 cell mutants selected by resistance to aphidicolin, a specific inhibitor of DNA polymerase-alpha, have been reported (Somatic Cell Genet., 7: 235-253, 1981). DNA polymerase-alpha was purified from mitochondria-free crude extracts of these mutants by sequential column chromatography using DEAE-cellulose and phosphocellulose. DNA polymerase-alpha purified from one of the mutants is 10-fold more resistant to aphidicolin than the same enzyme purified from the parental cells. Moreover, the apparent Km for dCTP is 1.0 +/- 0.4 microM for the mutant polymerase and 10 +/- 4 microM for the parental enzyme. These observed differences are in accord with the known competition between aphidicolin and dCTP, and provide a mechanism for the aphidicolin resistance of the mutant, i.e., the decrease in Km for dCTP. The elevated spontaneous and induced mutation rate exhibited by this mutant could be mediated by the alteration in DNA polymerase-alpha. With DNA replicating enzymes from a variety of sources, enhancement of mutagenesis has been demonstrated by alteration in precursor pools, damage to DNA templates, loss of nucleotide bases on DNA, metal ions that interact with nucleotide bases, and organic compounds that intercalate into DNA. The alterations of deoxynucleoside triphosphate pools also occur after treatment of animal cells with known mutagens. This observation may provide a new mechanism for mutagenesis by these agents independent of alterations in DNA.
Animals↗
Caffeine delays replication fork progression and enhances UV-induced homologous recombination in Chinese hamster cell lines.
The ability to bypass DNA lesions encountered during replication is important in order to maintain cell viability and avoid genomic instability. Exposure of mammalian cells to UV-irradiation induces the formation of DNA lesions that stall replication forks. In order to restore replication, different bypass mechanisms are operating, previously named post-replication repair. Translesion DNA synthesis is performed by low-fidelity polymerases, which can replicate across damaged sites. The nature of lesions and of polymerases involved influences the resulting frequency of mutations. Homologous recombination represents an alternative pathway for the rescue of stalled replication forks. Caffeine has long been recognized to influence post-replication repair, although the mechanism is not identified. Here, we found that caffeine delays the progress of replication forks in UV-irradiated Chinese hamster cells. The length of this enhanced delay was similar in wild-type cells and in cell deficient in either homologous recombination or nucleotide excision repair. Furthermore, caffeine attenuated the frequency of UV-induced mutations in the hprt gene, whereas the frequency of recombination, monitored in this same gene, was enhanced. These observations indicate that in cells exposed to UV-light, caffeine inhibits the rescue of stalled replication forks by translesion DNA synthesis, thereby causing a switch to bypass via homologous recombination. The biological consequence of the former pathway is mutations, while the latter results in chromosomal aberrations.
Animals↗
Inefficient proofreading and biased error rates during inaccurate DNA synthesis by a mutant derivative of Saccharomyces cerevisiae DNA polymerase delta.
DNA polymerase delta (pol delta) is a high fidelity eukaryotic enzyme that participates in DNA repair and is essential for DNA replication. Toward the goal of dissecting its multiple biological functions, here we describe the biochemical properties of Saccharomyces cerevisiae pol delta with a methionine replacing conserved leucine 612 at the polymerase active site. Compared with wild type pol delta, L612M pol delta has normal processivity and slightly higher polymerase specific activity. L612M pol delta also has normal 3' exonuclease activity, yet it is impaired in partitioning mismatches to the exonuclease active site, thereby reducing DNA synthesis fidelity. Error rates in vitro for L612M pol delta are elevated for both base substitutions and single base deletions but in a highly biased manner. For each of the six possible pairs of reciprocal mismatches that could arise during replication of complementary DNA strands to account for any particular base substitution in vivo (e.g. T-dGMP or A-dCMP for T to C transitions), L612M pol delta error rates are substantially higher for one mismatch than the other. These results provide a biochemical explanation for our observation, which confirms earlier genetic studies, that a haploid pol3-L612M S. cerevisiae strain has an elevated spontaneous mutation rate that is likely due to reduced replication fidelity in vivo.
Amino Acid Sequence↗
An overview of the mechanisms of mutagenesis and carcinogenesis.
Cancer is a genetic disease due to the accumulation of numerous mutations rendering the tumour cell insensitive to control by the local cellular environment and by the whole organism. Analysis of the frequency of appearance of human cancer as a function of age shows that between four and seven mutations in key genes are usually necessary to produce most human cancers. Interesting debates in the literature are concerned with the idea that normal mutation rates followed by selective advantage of mutated clones are enough to produce the numerous mutations found in human cancers. Alternatively, the mutator phenotype hypothesis is based on the idea that the normal mutation rates are insufficient to account for the multiple mutations found in tumours. It is, however, difficult not only to know this exact mutation frequency in cells but also to know the total number of cell divisions giving rise to a cancer. Therefore, during at least one step in the carcinogenic process, a mutator phenotype in target cells may occur due to mutations controlling the fidelity of DNA replication or DNA repair, the apoptosis pathways or the cell cycle checkpoint regulations. Among the multiple mutations found in human cancers such as gene amplification, chromosome alterations and translocations, point mutations are very important and the molecular mechanisms of their production are well documented. I will describe in detail the various mechanisms that a cell can use to produce point mutations due to lower fidelity in the DNA polymerisation step or to inefficient repair pathways. The presence of multiple mutations in human cancer is interesting not only in terms of understanding the carcinogenesis process in humans but also in eventually promoting strategies to decrease the efficiency of this process and to increase cancer therapy regimen.
Aging↗
A possible role for chromium(III) in genotoxicity.
Chromium is found in the environment in two major forms: reduced CrIII and CrVI, or chromate. Chromate, the most biologically active species, is readily taken up by living cells and reduced intracellularly, via reactive intermediates, to stable CrIII species. CrIII, the most abundant form of chromium in the environment, does not readily cross cell membranes and is relatively inactive in vivo. However, intracellular CrIII can react slowly with both nucleic acids and proteins and can be genotoxic. We have investigated the genotoxicity of CrIII in vitro using a DNA replication assay and in vivo by CaCl2-mediated transfection of chromium-treated DNA into Escherichia coli. When DNA replication was measured on a CrIII-treated template using purified DNA polymerases (either bacterial or mammalian), both the rate of DNA replication and the amount of incorporation per polymerase binding event (processivity) were greatly increased relative to controls. When transfected into E. coli, CrIII-treated M13mp2 bacteriophage DNA showed a dose-dependent increase in mutation frequency. These results suggest that CrIII alters the interaction between the DNA template and the polymerase such that the binding strength of the DNA polymerase is increased and the fidelity of DNA replication is decreased. These interactions may contribute to the mutagenicity of chromium ions in vivo and suggest that CrIII can contribute to chromium-mediated carcinogenesis.
Chromium↗
Replicative enzymes, DNA polymerase alpha (pol alpha), and in vitro ageing.
Normal cells in culture are used to investigate the underlying mechanisms of DNA synthesis because they retain regulatory characteristics of the in vivo replication machinery. During the last few years new studies have identified a number of genetic changes that occur during in vitro ageing, providing insight into the progressive decline in biological function that occurs during ageing. Maintaining genomic integrity in eukaryotic organisms requires precisely coordinated replication of the genome during mitosis, which is the most fundamental aspect of living cells. To achieve this coordinated replication, eukaryotic cells employ an ordered series of steps to form several key protein assemblies at origins of replication. Major progress has recently been made in identifying the enzymes, and other proteins, of DNA replication that are recruited to origin sites and the order in which they are recruited during the process of replication. More than 20 proteins, including DNA polymerases, have been identified as essential components that must be preassembled at replication origins for the initiation of DNA synthesis. Of the polymerases, DNA polymerase alpha-primase (pol alpha) is of particular importance since its function is fundamental to understanding the initiation mechanism of eukaryotic DNA replication. DNA must be replicated with high fidelity to ensure the accurate transfer of genetic information to progeny cells, and decreases in DNA pol alpha activity and fidelity, which are coordinated with cell cycle progression, have been shown to be important facets of a probable intrinsic cause of genetic alterations during in vitro ageing. This has led to the proposal that pol alpha activity and function is one of the crucial determinants in ageing. In this review we summarize the current state of knowledge of DNA pol alpha function in the regulation of DNA replication and focus in particular on its interactive tasks with other proteins during in vitro ageing.
Cell Cycle↗
Translesion synthesis DNA polymerases and control of genome stability.
Eukaryotic and prokaryotic genomes are replicated with amazingly high fidelity to assure faithful transmission of genetic information from one generation to the next. The accuracy of replication relies heavily on the ability of replicative DNA polymerases to efficiently select correct nucleotides for the polymerization reaction and excise mistakenly incorporated nucleotides using their intrinsic exonucleases. Cell also possess a variety of specialized DNA polymerases that help to overcome replication blocks when occasional unrepaired DNA lesions stall the replication machinery. The translesion synthesis (TLS) polymerases have an extremely low fidelity during copying undamaged DNA substrates, such that uncontrolled participation of these polymerases in DNA replication could present a threat to the genome stability. In this article, we discuss the properties of prokaryotic and eukaryotic TLS polymerases and their roles in modulating the rate of spontaneous and genotoxicant-induced mutations. We also review recent insights into the molecular mechanisms regulating the participation of error-prone TLS polymerases in the genome replication. Finally, we discuss the relationship between the functions of TLS polymerases and human disease.
DNA Damage↗
In vivo mutagenesis by Escherichia coli DNA polymerase I. Ile(709) in motif A functions in base selection.
The fidelity of DNA replication by Escherichia coli DNA polymerase I (pol I) was assessed in vivo using a reporter plasmid bearing a ColE1-type origin and an ochre codon in the beta-lactamase gene. We screened 53 single mutants within the region Val(700)-Arg(712) in the polymerase active-site motif A. Only replacement of Ile(709) yielded mutator polymerases, with substitution of Met, Asn, Phe, or Ala increasing the beta-lactamase reversion frequency 5-23-fold. Steady-state kinetic analysis of the I709F polymerase revealed reductions in apparent K(m) values for both insertion of non-complementary nucleotides and extension of mispaired primer termini. Abolishment of the 3'-5' exonuclease activity of wild-type pol I increased mutation frequency 4-fold, whereas the combination of I709F and lack of the 3'-5' exonuclease yielded a 400-fold increase. We conclude that accurate discrimination of the incoming nucleotide at the polymerase domain is more critical than exonucleolytic proofreading for the fidelity of pol I in vivo. Surprisingly, the I709F polymerase enhanced mutagenesis in chromosomal DNA, although the increase was 10-fold less than in plasmid DNA. Our findings indicate the feasibility of obtaining desired mutations by replicating a target gene at a specific locus in a plasmid under continuous selection pressure.
Amino Acid Motifs↗
The 3' to 5' exonuclease activity located in the DNA polymerase delta subunit of Saccharomyces cerevisiae is required for accurate replication.
In Saccharomyces cerevisiae, DNA polymerase delta (POLIII), the product of the CDC2 (POL3) gene, possesses, in its N-terminal half, the well conserved 3-domain 3' to 5' exonuclease site. Strains selectively mutagenized in this site display a mutator phenotype detected as a drastically increased spontaneous forward mutation rate to canavanine resistance or as an elevated reversion rate to lysine prototrophy. Assays on a partially purified extract of the mutant giving the largest mutator effect indicate that the 3' to 5' exonuclease activity is reduced below the detection limit whereas the DNA polymerizing activity has wild-type level. Therefore, our results provide experimental support for the hypothesis that the exonucleolytic proofreading activity associated with DNA polymerase delta resides on the DNA polymerase delta subunit and enhances the fidelity of DNA replication in yeast.
Amino Acid Sequence↗
High fidelity of homologous retroviral recombination in cell culture.
Genetic variation continues to be a major obstacle in the development of therapies and vaccines against retroviral infections and contributes extensively to viral pathogenesis and persistence. Recombination is one mechanism that increases retroviral variation by shuffling mutations from different genomes. Recent studies suggest that recombination not only shuffles the mutations but also generates them at high rates during reverse transcription. In contrast to these recent studies, this investigation shows that recombination does not generate mutations during recombination. A spleen necrosis virus (SNV)-based homologous recombination system was used to test the hypothesis that retroviral recombination is a high-fidelity process during replication of the virus in cell culture. The system consisted of a pair of SNV vectors expressing two drug resistance genes. The vectors were constructed so that cells containing recombinant proviruses could be selected by a double drug-resistant phenotype. Restriction enzyme digestion and agarose gel electrophoresis were used to map the location of recombination within 182 proviruses. Sequencing and single-strand conformation polymorphism techniques were then used to check for mutations within the recombinant proviruses. Since no mutations were detected among the 182 recombinants that were analyzed, homologous recombination is a high-fidelity process for retroviruses in cell culture.
Cells, Cultured↗
Real ribozymes suggest a relaxed error threshold.
The error threshold for replication, the critical copying fidelity below which the fittest genotype deterministically disappears, limits the length of the genome that can be maintained by selection. Primordial replication must have been error-prone, and so early replicators are thought to have been necessarily short. The error threshold also depends on the fitness landscape. In an RNA world, many neutral and compensatory mutations can raise the threshold, below which the functional phenotype, rather than a particular sequence, is still present. Here we show, on the basis of comparative analysis of two extensively mutagenized ribozymes, that with a copying fidelity of 0.999 per digit per replication the phenotypic error threshold rises well above 7,000 nucleotides, which permits the selective maintenance of a functionally rich riboorganism with a genome of more than 100 different genes, the size of a tRNA. This requires an order of magnitude of improvement in the accuracy of in vitro-generated polymerase ribozymes. Incidentally, this genome size coincides with that estimated for a minimal cell achieved by top-down analysis, omitting the genes dealing with translation.
Base Pairing↗
Nidovirales: evolving the largest RNA virus genome.
This review focuses on the monophyletic group of animal RNA viruses united in the order Nidovirales. The order includes the distantly related coronaviruses, toroviruses, and roniviruses, which possess the largest known RNA genomes (from 26 to 32kb) and will therefore be called "large" nidoviruses in this review. They are compared with their arterivirus cousins, which also belong to the Nidovirales despite having a much smaller genome (13-16kb). Common and unique features that have been identified for either large or all nidoviruses are outlined. These include the nidovirus genetic plan and genome diversity, the composition of the replicase machinery and virus particles, virus-specific accessory genes, the mechanisms of RNA and protein synthesis, and the origin and evolution of nidoviruses with small and large genomes. Nidoviruses employ single-stranded, polycistronic RNA genomes of positive polarity that direct the synthesis of the subunits of the replicative complex, including the RNA-dependent RNA polymerase and helicase. Replicase gene expression is under the principal control of a ribosomal frameshifting signal and a chymotrypsin-like protease, which is assisted by one or more papain-like proteases. A nested set of subgenomic RNAs is synthesized to express the 3'-proximal ORFs that encode most conserved structural proteins and, in some large nidoviruses, also diverse accessory proteins that may promote virus adaptation to specific hosts. The replicase machinery includes a set of RNA-processing enzymes some of which are unique for either all or large nidoviruses. The acquisition of these enzymes may have improved the low fidelity of RNA replication to allow genome expansion and give rise to the ancestors of small and, subsequently, large nidoviruses.
Evolution, Molecular↗
Exo+ proofreading polymerases mediate genetic analysis and its application in biomedical studies.
Polymerases with a proofreading function in their internal 3' to 5' exonuclease possess high fidelity for DNA replication both in vivo and in vitro. The obstacle facing Exo+ polymerases for single nucleotide polymorphism (SNP) detection could be bypassed by using primer-3'-termini modification. This hypothesis has been well tested using three types of modified allele specific primers with: 3' labeling, 3' to 5' exonuclease resistance, and 3' dehydroxylation. Accordingly, three new SNP assaying methods have been developed to carry out genome-wide genotyping, taking advantage of the enzymatic properties of Exo+ polymerases. These new mutation detection assays are widely adaptable to a variety of platforms, including multi-well plate and microarray technologies. Application of Exo+ polymerases to genetic analysis, including genotyping that is mostly relevant to pharma-cogenetics, high-fidelity gene expression profiling, rare mutation detection and mutation load assay, will help to accelerate the pace of personalized medicine. In this review paper, we will first introduce three new assays that we have recently developed, and then describe a number of their applications in pharmacogenetics and in other biomedical studies.
DNA Primers↗