The mutation rate and cancer

The selection of advantageous mutations underlies tumorigenesis. The growth of a tumor is therefore a form of evolution at the somatic level, in which the population is comprised of individual cells within the tumor. Models of tumorigenesis have considered the relative importance of mutation and selection. We show that selection is more important than an increased mutation rate in the growth of a tumor. Some cancers may acquire a “mutator phenotype,” probably leading to faster growth, but mutator phenotypes are not necessary for carcinogenesis.

Mutation rate varies among alleles at a microsatellite locus: Phylogenetic evidence

The understanding of the mutational mechanism that generates high levels of variation at microsatellite loci lags far behind the application of these genetic markers. A phylogenetic approach was developed to study the pattern and rate of mutations at a dinucleotide microsatellite locus tightly linked to HLA-DQB1 (DQCAR). A random Japanese population (n = 129) and a collection of multiethnic samples (n = 941) were typed at the DQB1 and DQCAR loci. The phylogeny of DQB1 alleles was then reconstructed and DQCAR alleles were superimposed onto the phylogeny. This approach allowed us to group DQCAR alleles that share a common ancestor. The results indicated that the DQCAR mutation rate varies drastically among alleles within this single microsatellite locus. Some DQCAR alleles never mutated during a long period of evolutionary time. Sequencing of representative DQCAR alleles showed that these alleles lost their ability to mutate because of nucleotide substitutions that shorten the length of uninterrupted CA repeat arrays; in contrast, all mutating alleles had relatively longer perfect CA repeat sequences.

The high spontaneous mutation rate: Is it a health risk?*

The human mutation rate for base substitutions is much higher in males than in females and increases with paternal age. This effect is mainly, if not entirely, due to the large number of cell divisions in the male germ line. The mutation-rate increase is considerably greater than expected if the mutation rate were simply proportional to the number of cell divisions. In contrast, those mutations that are small deletions or rearrangements do not show the paternal age effect. The observed increase with the age of the father in the incidence of children with different dominant mutations is variable, presumably the result of different mixtures of base substitutions and deletions. In Drosophila, the rate of mutations causing minor deleterious effects is estimated to be about one new mutation per zygote. Because of a larger number of genes and a much larger amount of DNA, the human rate is presumably higher. Recently, the Drosophila data have been reanalyzed and the mutation-rate estimate questioned, but I believe that the totality of evidence supports the original conclusion. The most reasonable way in which a species can cope with a high mutation rate is by quasi-truncation selection, whereby a number of mutant genes are eliminated by one “genetic death.”

Multilocus DNA fingerprinting reveals high rate of heritable genetic mutation in herring gulls nesting in an industrialized urban site.

Genotoxins, such as polycyclic aromatic compounds, are ubiquitous in urban and industrial environments. Our understanding of the role that these chemicals play in generating DNA sequence mutations is predominantly derived from laboratory studies with specific genotoxins or extracts of contaminants from environmental media. Most assays are not indicative of the germinal effects of exposure in situ to complex mixtures of common environmental mutagens. Using multilocus DNA fingerprinting, we found the mutation rate in herring gulls inhabiting a heavily industrialized urban harbor (Hamilton Harbour, Ontario) to be more than twice as high as three rural sites: Kent Island, Bay of Fundy; Chantry Island, Lake Huron; and Presqu'ile Provincial Park in Lake Ontario. Overall we found a mutation rate of 0.017 +/- 0.004 per offspring band in Hamilton, 0.006 +/- 0.002 at Kent Island, 0.002 +/- 0.002 from Chantry Island, and 0.004 +/- 0.002 from Presqu'ile Provincial Park. The mutation rate from the rural sites (pooled) was significantly lower than the rate observed in Hamilton Harbour (Fisher's exact test, two-tailed; P = 0.0006). These minisatellite DNA mutations may be important biomarkers for heritable genetic changes resulting from in situ exposure to environmental genotoxins in a free-living vertebrate species.

Mutation rates among RNA viruses

The rate of spontaneous mutation is a key parameter in modeling the genetic structure and evolution of populations. The impact of the accumulated load of mutations and the consequences of increasing the mutation rate are important in assessing the genetic health of populations. Mutation frequencies are among the more directly measurable population parameters, although the information needed to convert them into mutation rates is often lacking. A previous analysis of mutation rates in RNA viruses (specifically in riboviruses rather than retroviruses) was constrained by the quality and quantity of available measurements and by the lack of a specific theoretical framework for converting mutation frequencies into mutation rates in this group of organisms. Here, we describe a simple relation between ribovirus mutation frequencies and mutation rates, apply it to the best (albeit far from satisfactory) available data, and observe a central value for the mutation rate per genome per replication of μg ≈ 0.76. (The rate per round of cell infection is twice this value or about 1.5.) This value is so large, and ribovirus genomes are so informationally dense, that even a modest increase extinguishes the population.

Stage specificity, dose response, and doubling dose for mouse minisatellite germ-line mutation induced by acute radiation

Germ-line mutation induction at mouse minisatellite loci by acute irradiation with x-rays was studied at premeiotic and postmeiotic stages of spermatogenesis. An elevated paternal mutation rate was found after irradiation of premeiotic spermatogonia and stem cells, whereas the frequency of minisatellite mutation after postmeiotic irradiation of spermatids was similar to that in control litters. In contrast, paternal irradiation did not affect the maternal mutation rate. A linear dose–response curve for paternal mutation induced at premeiotic stages was found, with a doubling dose of 0.33 Gy, a value close to those obtained in mice after acute spermatogonia irradiation using other systems for mutation detection. High frequencies of spontaneous and induced mutations at minisatellite loci allow mutation induction to be evaluated at low doses of exposure in very small population samples, which currently makes minisatellite DNA the most powerful tool for monitoring radiation-induced germ-line mutation.

Genetic fidelity under harsh conditions: Analysis of spontaneous mutation in the thermoacidophilic archaeon Sulfolobus acidocaldarius

Microbes whose genomes are encoded by DNA and for which adequate information is available display similar genomic mutation rates (average 0.0034 mutations per chromosome replication, range 0.0025 to 0.0046). However, this value currently is based on only a few well characterized microbes reproducing within a narrow range of environmental conditions. In particular, no genomic mutation rate has been determined either for a microbe whose natural growth conditions may extensively damage DNA or for any member of the archaea, a prokaryotic lineage deeply diverged from both bacteria and eukaryotes. Both of these conditions are met by the extreme thermoacidophile Sulfolobus acidocaldarius. We determined the genomic mutation rate for this species when growing at pH 3.5 and 75°C based on the rate of forward mutation at the pyrE gene and the nucleotide changes identified in 101 independent mutants. The observed value of about 0.0018 extends the range of DNA-based microbes with rates close to the standard rate simultaneously to an archaeon and to an extremophile whose cytoplasmic pH and normal growth temperature greatly accelerate the spontaneous decomposition of DNA. The mutations include base pair substitutions (BPSs) and additions and deletions of various sizes, but the S. acidocaldarius spectrum differs from those of other DNA-based organisms in being relatively poor in BPSs. The paucity of BPSs cannot yet be explained by known properties of DNA replication or repair enzymes of Sulfolobus spp. It suggests, however, that molecular evolution per genome replication may proceed more slowly in S. acidocaldarius than in other DNA-based organisms examined to date.

Enhanced somatic mutation rates induced in stem cells of mice by low chronic exposure to ethylnitrosourea.

We have found that the somatic mutation rate at the Dlb-1 locus increases exponentially during low daily exposure to ethylnitrosourea over 4 months. This effect, enhanced mutagenesis, was not observed at a lacI transgene in the same tissue, although the two loci respond very similarly to acute doses. Since both mutations are neutral, the mutant frequency was expected to increase linearly with time in response to a constant mutagenic exposure, as it did for lacI. Enhanced mutagenesis does not result from an overall sensitization of the animals, since mice that had first been treated with a low daily dose for 90 days and then challenged with a large acute dose were not sensitized to the acute dose. Nor was the increased mutant frequency due to selection, since animals that were treated for 90 days and then left untreated for up to 60 days showed little change from the 90-day frequency. The effect is substantial: about 8 times as many Dlb-1 mutants were induced between 90 and 120 days as in the first 30 days. This resulted in a reverse dose rate effect such that 90 mg/kg induced more mutants when delivered at 1 mg/kg per day than at 3 mg/kg per day. We postulate that enhanced mutagenesis arises from increased stem cell proliferation and the preferential repair of transcribed genes. Enhanced mutagenesis may be important for risk evaluation, as the results show that chronic exposures can be more mutagenic than acute ones and raise the possibility of synergism between chemicals at low doses.

Modeling and optimization of populations subject to time-dependent mutation.

It has become clear that many organisms possess the ability to regulate their mutation rate in response to environmental conditions. So the question of finding an optimal mutation rate must be replaced by that of finding an optimal mutation schedule. We show that this task cannot be accomplished with standard population-dynamic models. We then develop a "hybrid" model for populations experiencing time-dependent mutation that treats population growth as deterministic but the time of first appearance of new variants as stochastic. We show that the hybrid model agrees well with a Monte Carlo simulation. From this model, we derive a deterministic approximation, a "threshold" model, that is similar to standard population dynamic models but differs in the initial rate of generation of new mutants. We use these techniques to model antibody affinity maturation by somatic hypermutation. We had previously shown that the optimal mutation schedule for the deterministic threshold model is phasic, with periods of mutation between intervals of mutation-free growth. To establish the validity of this schedule, we now show that the phasic schedule that optimizes the deterministic threshold model significantly improves upon the best constant-rate schedule for the hybrid and Monte Carlo models.

Increased mutation frequency of feline immunodeficiency virus lacking functional deoxyuridine-triphosphatase.

Feline immunodeficiency virus (FIV) encodes the enzyme deoxyuridine-triphosphatase (DU; EC 3.6.1.23) between the coding regions for reverse transcriptase and integrase in the pol gene. Here, we report the in vivo infection of cats with a DU- variant of the PPR strain of FIV and compare its growth properties and tissue distribution with those of wild-type FIV-PPR. The results reveal several important points: (i) DU- FIV is able to infect the cat, with kinetics similar to that observed with wild-type FIV; (ii) both wild-type and DU- FIV-infected specific-pathogen free cats mount a strong humoral antibody response which is able to limit the virus burden in both groups of animals; (iii) the virus burden is reduced in the DU- FIV-infected cats, particularly in tissues such as spleen and salivary gland; and (iv) the mutation frequency in DU- FIVs integrated in the DNA of primary macrophages after 9 months of infection is approximately 5-fold greater than the frequency observed in DU- FIV DNA integrated in T lymphocytes. Mutation rate with wild-type FIV remains the same in both cell types in vivo. The dominant mutations seen in macrophages with DU- FIV are G-->A base changes, consistent with an increased misincorporation of deoxyuridine into viral DNA of DU- FIVs during reverse transcription. Because this enzyme is absent from human immunodeficiency virus type 1 and other primate lentiviruses, virus replication in cell environments with low DU activity may lead to increased mutation and contribute to the rapid expansion of the viral repertoire.

The universal ancestor

A genetic annealing model for the universal ancestor of all extant life is presented; the name of the model derives from its resemblance to physical annealing. The scenario pictured starts when “genetic temperatures” were very high, cellular entities (progenotes) were very simple, and information processing systems were inaccurate. Initially, both mutation rate and lateral gene transfer levels were elevated. The latter was pandemic and pervasive to the extent that it, not vertical inheritance, defined the evolutionary dynamic. As increasingly complex and precise biological structures and processes evolved, both the mutation rate and the scope and level of lateral gene transfer, i.e., evolutionary temperature, dropped, and the evolutionary dynamic gradually became that characteristic of modern cells. The various subsystems of the cell “crystallized,” i.e., became refractory to lateral gene transfer, at different stages of “cooling,” with the translation apparatus probably crystallizing first. Organismal lineages, and so organisms as we know them, did not exist at these early stages. The universal phylogenetic tree, therefore, is not an organismal tree at its base but gradually becomes one as its peripheral branchings emerge. The universal ancestor is not a discrete entity. It is, rather, a diverse community of cells that survives and evolves as a biological unit. This communal ancestor has a physical history but not a genealogical one. Over time, this ancestor refined into a smaller number of increasingly complex cell types with the ancestors of the three primary groupings of organisms arising as a result.

Calibrating bacterial evolution

Attempts to calibrate bacterial evolution have relied on the assumption that rates of molecular sequence divergence in bacteria are similar to those of higher eukaryotes, or to those of the few bacterial taxa for which ancestors can be reliably dated from ecological or geological evidence. Despite similarities in the substitution rates estimated for some lineages, comparisons of the relative rates of evolution at different classes of nucleotide sites indicate no basis for their universal application to all bacteria. However, there is evidence that bacteria have a constant genome-wide mutation rate on an evolutionary time scale but that this rate differs dramatically from the rate estimated by experimental methods.

A deletion within the murine Werner syndrome helicase induces sensitivity to inhibitors of topoisomerase and loss of cellular proliferative capacity

Werner syndrome (WS) is an autosomal recessive disorder characterized by genomic instability and the premature onset of a number of age-related diseases. The gene responsible for WS encodes a member of the RecQ-like subfamily of DNA helicases. Here we show that its murine homologue maps to murine chromosome 8 in a region syntenic with the human WRN gene. We have deleted a segment of this gene and created Wrn-deficient embryonic stem (ES) cells and WS mice. While displaying reduced embryonic survival, live-born WS mice otherwise appear normal during their first year of life. Nonetheless, although several DNA repair systems are apparently intact in homozygous WS ES cells, such cells display a higher mutation rate and are significantly more sensitive to topoisomerase inhibitors (especially camptothecin) than are wild-type ES cells. Furthermore, mouse embryo fibroblasts derived from homozygous WS embryos show premature loss of proliferative capacity. At the molecular level, wild-type, but not mutant, WS protein copurifies through a series of centrifugation and chromatography steps with a multiprotein DNA replication complex.

Accumulation of premutagenic DNA lesions in mice defective in removal of oxidative base damage

DNA damage generated by oxidant byproducts of cellular metabolism has been proposed as a key factor in cancer and aging. Oxygen free radicals cause predominantly base damage in DNA, and the most frequent mutagenic base lesion is 7,8-dihydro-8-oxoguanine (8-oxoG). This altered base can pair with A as well as C residues, leading to a greatly increased frequency of spontaneous G·C→T·A transversion mutations in repair-deficient bacterial and yeast cells. Eukaryotic cells use a specific DNA glycosylase, the product of the OGG1 gene, to excise 8-oxoG from DNA. To assess the role of the mammalian enzyme in repair of DNA damage and prevention of carcinogenesis, we have generated homozygous ogg1−/− null mice. These animals are viable but accumulate abnormal levels of 8-oxoG in their genomes. Despite this increase in potentially miscoding DNA lesions, OGG1-deficient mice exhibit only a moderately, but significantly, elevated spontaneous mutation rate in nonproliferative tissues, do not develop malignancies, and show no marked pathological changes. Extracts of ogg1 null mouse tissues cannot excise the damaged base, but there is significant slow removal in vivo from proliferating cells. These findings suggest that in the absence of the DNA glycosylase, and in apparent contrast to bacterial and yeast cells, an alternative repair pathway functions to minimize the effects of an increased load of 8-oxoG in the genome and maintain a low endogenous mutation frequency.

Spontaneous mutations recovered as mosaics in the mouse specific-locus test

The specific-locus test (SLT) detects new mutants among mice heterozygous for seven recessive visible markers. Spontaneous mutations can be manifested not only as singleton whole-body mutants in controls (for which we report new data), but as mosaics—either visible (manifesting mottled coat color) in the scored generation (G2) or masked, among the wild-type parental generation (G1). Masked G1 mosaics reveal themselves by producing clusters of whole-body mutants in G2. We provide evidence that most, if not all, mosaics detected in the SLT (both radiation and control progenies) result from a single-strand spontaneous mutation subsequent to the last premeiotic mitosis and before the first postmeiotic one of a parental genome—the “perigametic interval.” Such events in the genomes of the G1 and G0 result, respectively, in visible and masked 50:50 mosaics. Per cell cycle, the spontaneous mutation rate in the perigametic interval is much higher than that in pregamete mitotic divisions. A clearly different locus spectrum further supports the hypothesis of different origin, and casts further doubt on the validity of the doubling-dose risk-estimation method. Because mosaics cannot have arisen in mitotic germ cells, and are not induced by radiation exposure in the perigametic interval, they should not be included in calculations of radiation-induced germ-line mutation rates. For per-generation calculations, inclusion of mosaics yields a spontaneous frequency 1.7 times that calculated from singletons alone for mutations contributed by males; including both sexes, the multiple is 2.2.