Theoretical Study of near Neutrality. I. Heterozygosity and Rate of Mutant Substitution

In order to clarify the nature of "near neutrality" in molecular evolution and polymorphism, extensive simulation studies were performed. Selection coefficients of new mutations are assumed to be small so that both random genetic drift and selection contribute to determining the behavior of mutants. The model also incorporates normally distributed spatial fluctuation of selection coefficients. If the system starts from "average neutrality," it will move to a better adapted state, and most new mutations will become "slightly deleterious." Monte Carlo simulations have indicated that such adaptation is attained, but that the rate of such "progress" is very low for weak selection. In general, the larger the population size, the more effective the selection becomes. Also, as selection becomes weaker, the behavior of the mutants approaches that of completely neutral genes. Thus, the weaker the selection, the smaller is the effect of population size on mutant dynamics. Increase of heterozygosity with population size is very pronounced for subdivided populations. The significance of these results is discussed in relation to various observed facts on molecular evolution and polymorphism, such as generation-time dependency and overdispersion of the molecular clock, or contrasting patterns of DNA and protein polymorphism among some closely related species.     

DNA损伤修复机制——解读2015年诺贝尔化学奖

Tomas Lindahl, Paul Modrich和Aziz Sancar三位科学家因发现“DNA损伤修复机制”获得了2015年诺贝尔化学奖．Lindahl首次发现Escherichia Coli中参与碱基切除修复的第一个蛋白质--尿嘧啶 DNA糖基化酶（UNG）; Modrich重建了错配修复的体外系统,从大肠杆菌到哺乳动物深入探究了错配修复的机制; Sancar利用纯化的UvrA、UvrB、UvrC重建了核苷酸切除修复的关键步骤,阐述了核苷酸切除修复的分子机制．DNA损伤是由生物所处体外环境和体内因素共同导致的,面对不同种类的损伤,机体启动多种不同的修复机制修复损伤,保护基因组稳定性．这些修复机制包括：光修复(light repairing);核苷酸切除修复（nucleotide excision repair, NER）;碱基切除修复（base excision repair, BER）;错配修复（mismatch repair, MMR）;以及DNA双链断裂修复（DNA double strand breaks repair, DSBR）．其中DNA双链断裂修复又分同源重组（homologous recombination, HR）和非同源末端连接（non homologous end joining, NHEJ）两种方式．本文将对上述几种修复的机制进行总结与讨论．     

A Genetic Analysis of Primary Products of Bacteriophage Lambda Recombination

Primary products of bacteriophage lambda recombination that display heterozygosity as a consequence of the presence of regions of heteroduplex DNA are rare in standard lambda crosses. Phage manifesting heterozygosity at a given allele are evident when recombinants, emerging from a cross, are selected for an exchange in a neighboring interval. We show that the abundance of such heterozygotes can be increased 10- to 20-fold by selection on an E. coli indicator that is defective in methyl-directed mismatch repair (mutL). Thus, the activity of the methyl-directed mismatch repair system is, at least in part, responsible for the low frequency of detectably heterozygous phage emerging from a standard cross. In a mutL indicator, many primary products of recombination are replicated without the intervention of mismatch repair.--The products of a six-factor phage cross have been plated on a mutL indicator allowing visual detection of those phage products heterozygous for one of the allelic pairs, cI. By genetic analysis, we show that the heteroduplex regions of these primary products of recombination are on the average about 4 kb in length and can include as much as half of the lambda genome.     

Factors affecting levels of genetic diversity in natural populations.

Genetic variability is the clay of evolution, providing the base material on which adaptation and speciation depend. It is often assumed that most interspecific differences in variability are due primarily to population size effects, with bottlenecked populations carrying less variability than those of stable size. However, we show that population bottlenecks are unlikely to be the only factor, even in classic case studies such as the northern elephant seal and the cheetah, where genetic polymorphism is virtually absent. Instead, we suggest that the low levels of variability observed in endangered populations are more likely to result from a combination of publication biases, which tend to inflate the level of variability which is considered ''normal'', and inbreeding effects, which may hasten loss of variability due to drift. To account for species with large population sizes but low variability we advance three hypotheses. First, it is known that certain metapopulation structures can result in effective population sizes far below the census size. Second, there is increasing evidence that heterozygous sites mutate more frequently than equivalent homozygous sites, plausibly because mismatch repair between homologous chromosomes during meiosis provides extra opportunities to mutate. Such a mechanism would undermine the simple relationship between heterozygosity and effective population size. Third, the fact that related species that differ greatly in variability implies that large amounts of variability can be gained or lost rapidly. We argue that such cases are best explained by rapid loss through a genome-wide selective sweep, and suggest a mechanism by which this could come about, based on forced changes to a control gene inducing coevolution in the genes it controls. Our model, based on meiotic drive in mammals, but easily extended to other systems, would tend to facilitate population isolation by generating molecular incompatabilities. Circumstances can even be envisioned in which the process could provide intrinsic impetus to speciation.     

X-ray-induced DNA double-strand breaks in mouse l1210 cells: a new computational method for analyzing neutral filter elution data

The aim of this article is to present a method for studying the shape of the dose and repair responses for X-ray-induced double-strand breaks (DSBs) as measured by neutral filter elution (NFE). The approach is closely related to a method we developed for the use of specific molecular size markers and used for determination of the absolute number of randomly distributed radiation-induced DSBs by pulsed-field gel electrophoresis (PFGE). Mouse leukemia L1210 cells were X-irradiated with 0-50 Gy. Samples were then evaluated both with PFGE and with NFE. Assuming that with both migration (PFGE) and elution (NFE), a heterogeneous population of double-stranded DNA fragments will start with the smallest fragments and proceed with increasingly larger fragments, it is possible to match the migration behavior of fractions of fragments smaller than a certain size to the fraction eluted at a specific time. This assumption does not exclude the possibility of DNA being sheared in the NFE filter. The yield, as determined by the size markers in PFGE, was used to find the corresponding elution times in the NFE experiment. These experimentally used elution times could then reversely be interpreted as size markers which finally were used to calculate DSBs/Mbp as a function of X-ray dose. The resulting lines were almost straight. The data were also plotted as relative elution and showed that, as expected, the dose response then appears with a more pronounced sigmoid shape.     

A neutral model with fluctuating population size and its effective size

We consider a diffusion model with neutral alleles whose population size is fluctuating randomly. For this model, the effects of fluctuation of population size on the effective size are investigated. The effective size defined by the equilibrium average heterozygosity is larger than the harmonic mean of population size but smaller than the arithmetic mean of population size. To see explicitly the effects of fluctuation of population size on the effective size, we investigate a special case where population size fluctuates between two distinct states. In some cases, the effective size is very different from the harmonic mean. For this concrete model, we also obtain the stationary distribution of the average heterozygosity. Asymptotic behavior of the effective size is obtained when the population size is large and/or autocorrelation of the fluctuation is weak or strong.     

Inefficient mismatch repair: genetic defects and down regulation

The mismatch repair system is involved in the maintenance of genomic integrity by editing DNA replication and recombination. However, although most mutations are neutral or deleterious, a mutator phenotype due to an inefficient mismatch repair may generate advantageous variants and may therefore be selected for. We review the evidence for inefficient mismatch repair due either to genetic defects in mismatch repair genes or to physiological conditions. Among natural isolates ofEscherichia coli andSalmonella enterica, about 1% are mutator bacteria, mostly deficient in mismatch repair (most of them defective in themutS gene). Characterization of mutators derived from laboratory strains led also to the isolation of mismatch repair mutants in which the most frequently found defects are inmutL andmutS. The correlation of the size of the antimutator genes with the frequency of their defective alleles amongE. coli andSalmonella strains reveals thatmutU mutants are underrepresented. Analysis of the progeny of a defined M13 phage heteroduplex DNA transfected intoE. coli cells shows that mismatch repair efficiency progressively decreases from the end of the exponential growth in K-12 and is variable among natural isolates. Implications of this defective mismatch repair activity for evolution and tumorigenesis will be discussed.     

The role of DNA double-strand breaks in spontaneous homologous recombination in S. cerevisiae

Homologous recombination (HR) is a source of genomic instability and the loss of heterozygosity in mitotic cells. Since these events pose a severe health risk, it is important to understand the molecular events that cause spontaneous HR. In eukaryotes, high levels of HR are a normal feature of meiosis and result from the induction of a large number of DNA double-strand breaks (DSBs). By analogy, it is generally believed that the rare spontaneous mitotic HR events are due to repair of DNA DSBs that accidentally occur during mitotic growth. Here we provide the first direct evidence that most spontaneous mitotic HR in Saccharomyces cerevisiae is initiated by DNA lesions other than DSBs. Specifically, we describe a class of rad52 mutants that are fully proficient in inter- and intra-chromosomal mitotic HR, yet at the same time fail to repair DNA DSBs. The conclusions are drawn from genetic analyses, evaluation of the consequences of DSB repair failure at the DNA level, and examination of the cellular re-localization of Rad51 and mutant Rad52 proteins after introduction of specific DSBs. In further support of our conclusions, we show that, as in wild-type strains, UV-irradiation induces HR in these rad52 mutants, supporting the view that DNA nicks and single-stranded gaps, rather than DSBs, are major sources of spontaneous HR in mitotic yeast cells.     

CYS3, a hotspot of meiotic recombination in Saccharomyces cerevisiae. Effects of heterozygosity and mismatch repair functions on gene conversion and recombination intermediates

We have examined meiotic recombination at the CYS3 locus. Genetic analysis indicates that CYS3 is a hotspot of meiotic gene conversion, with a putative 5'-3' polarity gradient of conversion frequencies. This gradient is relieved in the presence of msh2 and pms1 mutations, indicating an involvement of mismatch repair functions in meiotic recombination. To investigate the role of mismatch repair proteins in meiotic recombination, we performed a physical analysis of meiotic DNA in wild-type and msh2 pms1 strains in the presence or absence of allelic differences at CYS3. Neither the mutations in CYS3 nor the absence of mismatch repair functions affects the frequency and distribution of nearby recombination-initiating DNA double-strand breaks (DSBs). Processing of DSBs is also similar in msh2 pms1 and wild-type strains. We conclude that mismatch repair functions do not control the distribution of meiotic gene conversion events at the initiating steps. In the MSH2 PMS1 background, strains heteroallelic for frameshift mutations in CYS3 exhibit a frequency of gene conversion greater than that observed for either marker alone. Physical analysis revealed no modification in the formation of DSBs, suggesting that this marker effect results from subsequent processing events that are not yet understood.     

Genetic parallelism of protein polymorphism in nature: ecological test of the neutral theory of molecular evolution

We have conducted an ecological test of protein polymorphism in 13 unrelated genera of plants, invertebrates and vertebrates, involving 21 species, 142 populations and 5474 individuals. Each was tested, on average, for 27 enzymatic gene loci. These species varied in population size and structure, life histories and biogeographical origins, but they largely share a geographically short (260 km) and ecologically stressful gradient of increasing aridity in Israel, both eastward and (mainly) southward. We found genetic parallelism across most taxa, and most loci. Observed average heterozygosity, H , and gene diversity, He , were positively and overall significantly correlated with rainfall variation. This result corroborates the environmental theory of genetic diversity, primarily the niche-width variation hypothesis in both space and time. Our results are inconsistent with the neutral theory of molecular evolution and suggest that natural selection appears to be an important differentiating evolutionary force at the protein level.     

Evaluation of DNA double strand breaks repair efficiency in head and neck cancer

Head and neck cancers (head and neck squamous cell carcinomas [HNSCC]) are a heterogeneous group of neoplasms with varying presenting symptoms, treatment, and expected outcome. There is a need to find an effective way of its treatment at the molecular level. Thus, we should identify the mechanism of cancer cell response to damaging agents' activity, especially at DNA level. Our major goal was to evaluate the efficacy of DNA double strand breaks (DSBs) repair in HTB-43 and SCC-25 cancer cell lines as well as lymphocytes taken from HNSCC patients and healthy donors. The DNA repair efficiency was measured by neutral comet assay as well as extrachromosomal assay for DNA DSBs repair (TAK assay). We determined the levels of two main pathways of DNA DSBs-nonhomologous end joining (NHEJ) and homologous recombination repair (HRR). Neutral comet assay was used for evaluation of DNA DSBs repair after treatment with genotoxic agents. DNA DSBs induced by gamma radiation were repaired slower in lymphocytes from HNSCC patients than in lymphocytes from healthy controls. HTB-43 and SCC-25 cancer cell lines have higher efficacy of NHEJ and HRR than lymphocytes taken from patients as well as control subjects. Our results confirm the necessity of further studies on the mechanisms of DNA DSBs repair to provide insight into the molecular basis of head and neck cancer, which will allow us to improve methods of HNSCC treatment.     

The repair of double-strand breaks in plants: mechanisms and consequences for genome evolution

The efficient repair of double-strand breaks (DSBs) in genomic DNA is important for the survival of all organisms. In recent years, basic mechanisms of DSB repair in somatic plant cells have been elucidated. DSBs are mainly repaired by non-homologous end-joining (NHEJ). The repair can be associated with deletions, but also insertions due to copying genomic sequences from elsewhere into the break. Species-specific differences of NHEJ have been reported and an inverse correlation of deletion size to genome size has been postulated, indicating that NHEJ might contribute significantly to evolution of genome size. DSB repair by homologous recombination (HR) might also influence genome organization. Whereas homology present in an allelic or an ectopic position is hardly used for repair, the use of homologous sequences in close proximity to the break is frequent. A 'single-strand annealing' mechanism that leads to sequence deletions between direct repeats is particularly efficient. This might explain the accumulation of single long terminal repeats of retroelements in cereal genomes. The conservative 'synthesis-dependent strand annealing' mechanism, resulting in conversions without crossovers is also prominent and seems to be significant for the evolution of tandemly arranged gene families such as resistance genes. Induction of DSBs could be used as a means for the controlled manipulation of plant genomes in an analogous way for the use of marker gene excision and site-specific integration.     

The nearly neutral and selection theories of molecular evolution under the fisher geometrical framework: substitution rate, population size, and complexity

The general theories of molecular evolution depend on relatively arbitrary assumptions about the relative distribution and rate of advantageous, deleterious, neutral, and nearly neutral mutations. The Fisher geometrical model (FGM) has been used to make distributions of mutations biologically interpretable. We explored an FGM-based molecular model to represent molecular evolutionary processes typically studied by nearly neutral and selection models, but in which distributions and relative rates of mutations with different selection coefficients are a consequence of biologically interpretable parameters, such as the average size of the phenotypic effect of mutations and the number of traits (complexity) of organisms. A variant of the FGM-based model that we called the static regime (SR) represents evolution as a nearly neutral process in which substitution rates are determined by a dynamic substitution process in which the population's phenotype remains around a suboptimum equilibrium fitness produced by a balance between slightly deleterious and slightly advantageous compensatory substitutions. As in previous nearly neutral models, the SR predicts a negative relationship between molecular evolutionary rate and population size; however, SR does not have the unrealistic properties of previous nearly neutral models such as the narrow window of selection strengths in which they work. In addition, the SR suggests that compensatory mutations cannot explain the high rate of fixations driven by positive selection currently found in DNA sequences, contrary to what has been previously suggested. We also developed a generalization of SR in which the optimum phenotype can change stochastically due to environmental or physiological shifts, which we called the variable regime (VR). VR models evolution as an interplay between adaptive processes and nearly neutral steady-state processes. When strong environmental fluctuations are incorporated, the process becomes a selection model in which evolutionary rate does not depend on population size, but is critically dependent on the complexity of organisms and mutation size. For SR as well as VR we found that key parameters of molecular evolution are linked by biological factors, and we showed that they cannot be fixed independently by arbitrary criteria, as has usually been assumed in previous molecular evolutionary models.     

The Variance of the Number of Loci Having a Given Gene Frequency

Considering a random mating population of finite size, the variance of the number of loci having a given gene frequency was derived under the assumption of a steady flux of mutations. The variance of average heterozygosity among populations was also derived under the same assumption. It was shown that these variances are proportional to the population size if the mutants are selectively neutral, and they are inversely proportional to the selection coefficient if the mutants are selectively advantageous and additive in their fitness.     

A Study on a Nearly Neutral Mutation Model in Finite Populations

As a nearly neutral mutation model, the house-of-cards model is studied in finite populations using computer simulations. The distribution of the mutant effect is assumed to be normal. The behavior is mainly determined by the product of the population size, N, and the standard deviation, sigma, of the distribution of the mutant effect. If 4N sigma is large compared to one, a few advantageous mutants are quickly fixed in early generations. Then most mutation becomes deleterious and very slow increase of the average selection coefficient follows. It takes very long for the population to reach the equilibrium state. Substitutions of alleles occur very infrequently in the later stage. If 4N sigma is the order of one or less, the behavior is qualitatively similar to that of the strict neutral case. Gradual increase of the average selection coefficient occurs and in generations of several times the inverse of the mutation rate the population almost reaches the equilibrium state. Both advantageous and neutral (including slightly deleterious) mutations are fixed. Except in the early stage, an increase of the standard deviation of the distribution of the mutant effect decreases the average heterozygosity. The substitution rate is reduced as 4N sigma is increased. Three tests of neutrality, one using the relationship between the average and the variance of heterozygosity, another using the relationship between the average heterozygosity and the average number of substitutions and Watterson's homozygosity test are applied to the consequences of the present model. It is found that deviation from the neutral expectation becomes apparent only when 4N sigma is more than two. Also a simple approximation for the model is developed which works well when the mutation rate is very small.     

Rare variant alleles in the light of the neutral theory

Based on the neutral theory of molecular evolution and polymorphism, and particularly assuming "the model of infinite alleles," a method is proposed which enables us to estimate the fraction of selectively neutral alleles (denoted by Pneut) among newly arisen mutations. It makes use of data on the distribution of rare variant alleles in large samples together with information on the average heterozygosity. The formula proposed is Pneut = [He/(1-He)] [loge(2nq)/n alpha (x less than q)], where n alpha(x less than q) is the average number of rare alleles per locus whose frequency, x, is less than q; n is the average sample size used to count rare alleles; He is the average heterozygosity per locus; and q is a small preassigned number such as q = 0.01. The method was applied to observations on enzyme and other protein loci in plaice, humans (European and Amerindian), Japanese monkeys, and fruit flies. Estimates obtained for them range from 0.064 to 0.21 with the mean and standard error Pneut = 0.14 +/- 0.06. It was pointed out that these estimates are consistent with the corresponding estimate Pneut(Hb) = 0.14 obtained independently based on the neutral theory and using data on the evolutionary rate of nucleotide substitutions in globin pseudogenes together with those in the normal globins.      

Effects of the shape of distribution of mutant effect in nearly neutral mutation models

Models of the theory of nearly neutral mutation incorporate a continuous distribution of mutation effects in contrast to the theory of purely neutral mutation which allows no mutations with intermediate effects. Previous studies of one such model, namely the house-of-cards mutation model, assumed normal distribution of mutation effect. Here I study the house-of-cards mutation model in random-mating finite populations using the weak-mutation approximation, paying attention to the effects of the distribution of mutant effects. The average selection coefficient, substitution rate and average heterozygosity in the equilibrium and transient states were studied mainly by computer simulation. The main findings are: (i) Very rapid decrease of the substitution rate and very slow approach to equilibrium as selection becomes stronger are characteristics of assuming normal distribution of mutant effect. If the right tail of the mutation distribution decays more rapidly than that of the normal distribution, the decrease of substitution rate becomes slower and equilibrium is achieved more quickly. (ii) The dispersion index becomes smaller or larger than 1 depending on the time and the intensity of selection, (iii) LetN be the population size. When selection is strong the ratio of 4N times the substitution rate to the average heterozygosity, which is expected to be 1 under neutrality, is larger than 1 in earlier generations but becomes less than 1 in later generations. These findings show the importance of the distribution of mutant effect and time in determination of the behaviour of various statistics frequently used in the study of molecular evolution.     

Species-specific double-strand break repair and genome evolution in plants

Even closely related eukaryotic species may differ drastically in genome size. While insertion of retroelements represents a major source of genome enlargement, the mechanism mediating species- specific deletions is fairly obscure. We analyzed the formation of deletions during double-strand break (DSB) repair in Arabidopsis thaliana and tobacco, two dicotyledonous plant species differing >20-fold in genome size. DSBs were induced by the rare cutting restriction endonuclease I-SCE:I and deletions were identified by loss of function of a negative selectable marker gene containing an I-SCE:I site. Whereas the partial use of micro-homologies in junction formation was similar in both species, in tobacco 40% of the deletions were accompanied by insertions. No insertions could be detected in Arabidopsis , where larger deletions were more frequent, indicating a putative inverse correlation between genome size and the average length of deletions. Such a correlation has been postulated before by a theoretical study on the evolution of related insect genomes and our study now identifies a possible molecular cause for the phenomenon, indicating that species-specific differences in DSB repair might indeed influence genome evolution.     

The role of population size in molecular evolution.

The results of a computer simulation study of the role of population size in population genetical models of molecular evolution are presented. If the mutation rate and strength of selection are held fixed and the population size increased, the eight models examined fall into three domains based on their rates of substitution. In the Ohta domain, the rate of substitution decreases with increasing population size; in the Kimura domain, the rate of substitution remains close to the mutation rate; in the Darwin domain, the rate of substitution increases without bound. In the Kimura and Darwin domains, the rate of substitution is much less sensitive to the population size than suggested by two-allele theories. Remarkably, the overdominance model converges to the neutral model with increasing N. The variation at a neutral locus linked to a selected locus is found to be insensitive to the population size for certain models of selection. A selected locus can actually cause the rate of substitution of deleterious alleles at a linked locus to increase with increasing population size. These unexpected results illustrate that intuition based on two-allele theory is often misleading.