How important is DNA replication for mutagenesis?

Rates of mutation and substitution in mammals are generally greater in the germ lines of males. This is usually explained as resulting from the larger number of germ cell divisions during spermatogenesis compared with oogenesis, with the assumption made that mutations occur primarily during DNA replication. However, the rate of cell division is not the only difference between male and female germ lines, and mechanisms are known that can give rise to mutations independently of DNA replication. We investigate the possibility that there are other causes of male-biased mutation. First, we show that patterns of variation at approximately 5,200 short tandem repeat (STR) loci indicate a higher mutation rate in males. We estimate a ratio of male-to-female mutation rates of approximately 1.9. This is significantly greater than 1 and supports a greater rate of mutation in males, affecting the evolution of these loci. Second, we show that there are chromosome-specific patterns of nucleotide and dinucleotide composition in mammals that have been shaped by mutation at CpG dinucleotides. Comparable patterns occur in birds. In mammals, male germ lines are more methylated than female germ lines, and these patterns indicate that differential methylation has played a role in male-biased vertebrate evolution. However, estimates of male mutation bias obtained from both classes of mutation are substantially lower than estimates of cell division bias from anatomical data. This discrepancy, along with published data indicating slipped-strand mispairing arising at STR loci in nonreplicating DNA, suggests that a substantial percentage of mutation may occur in nonreplicating DNA.     

Mammalian Male Mutation Bias: Impacts of Generation Time and Regional Variation in Substitution Rates

In mammals, males undergo a greater number of germline cell divisions compared with females. Thus, the male germline accumulates more DNA replication errors, which result in male mutation bias—a higher mutation rate for males than for females. The phenomenon of male mutation bias has been investigated mostly for rodents and primates, however, it has not been studied in detail for other mammalian orders. Here we sequenced and analyzed five introns of three genes (DBX/DBY, UTX/UTY, and ZFX/ZFY) homologous between X and Y chromosomes in several species of perissodactyls (horses and rhinos) and of primates. Male mutation bias was evident: substitution rate was higher for a Y chromosome intron than for its X chromosome homologue for all five intron pairs studied. Substitution rates varied regionally among introns sequenced on the same chromosome and this variation influenced male mutation bias inferred from each intron pair. Interestingly, we observed a positive correlation in substitution rates between homologous X and homologous Y introns as well as between orthologous primate and perissodactyl introns. The male-to-female mutation rate ratio estimated from concatenated sequences of five perissodactyl introns was 3.88 (95% CI = 2.90–6.07). Using the data generated here and estimates available in the literature, we compared male mutation bias among several mammalian orders. We conclude that male mutation bias is significantly higher for organisms with long generation times (primates, perissodactyls, and felids) than for organisms with short generation times (e.g., rodents) since the former undergo a greater number of male germline cell divisions. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Deborah Charlesworth]     

Sex-specific mutation rates in salmonid fish

If germline mutations arise because of replication errors, the mutation rate may differ between males and females given that they differ in their number of germ cell divisions. As males of many higher organisms produce more gametes than females, this has led to the idea of "male-driven evolution." The extent of such male bias to the mutation rate is currently debated. For human some recent data suggest a very low bias, at a factor 1.7 only, while other approaches have given values of alpha(m) (the male-to-female mutation rate ratio) of 5, which is more close to what might be expected from male and female germ cell biology. Comparative analyses of sex-specific mutation rates in other organisms may be necessary for understanding the generality of an effect of sex and the number of germline DNA replications on the mutation rate. In this study we estimate for the first time sex-specific mutation rates in fish. Comparing the intronic substitution rates of the autosomal GH- 2 gene and its duplicated Y-linked and male-specific copy GH- 2Y (447-468 bp of each gene), we estimate alpha(m) to be 5.35-6.60 in salmonid fish of the genus Oncorhynchus. To the observations previously made among mammals and birds, this adds evidence from another class of vertebrates showing that a majority of mutations are of paternal origin. This would suggest that replication errors play a major role for the generation of new mutations.     

Characteristics, causes and evolutionary consequences of male-biased mutation

Mutation has traditionally been considered a random process, but this paradigm is challenged by recent evidence of divergence rate heterogeneity in different genomic regions. One facet of mutation rate variation is the propensity for genetic change to correlate with the number of germ cell divisions, reflecting the replication-dependent origin of many mutations. Haldane was the first to connect this association of replication and mutation to the difference in the number of cell divisions in oogenesis (low) and spermatogenesis (usually high), and the resulting sex difference in the rate of mutation. The concept of male-biased mutation has been thoroughly analysed in recent years using an evolutionary approach, in which sequence divergence of autosomes and/or sex chromosomes are compared to allow inference about the relative contribution of mothers and fathers in the accumulation of mutations. For instance, assuming that a neutral sequence is analysed, that rate heterogeneity owing to other factors is cancelled out by the investigation of many loci and that the effect of ancestral polymorphism is properly taken into account, the male-to-female mutation rate ratio, alpham, can be solved from the observed difference in rate of X and Y chromosome divergence. The male mutation bias is positively correlated with the relative excess of cell divisions in the male compared to the female germ line, as evidenced by a generation time effect: in mammals, alpham is estimated at approximately 4-6 in primates, approximately 3 in carnivores and approximately 2 in small rodents. Another life-history correlate is sexual selection: when there is intense sperm competition among males, increased sperm production will be associated with a larger number of mitotic cell divisions in spermatogenesis and hence an increase in alpham. Male-biased mutation has implications for important aspects of evolutionary biology such as mate choice in relation to mutation load, sexual selection and the maintenance of genetic diversity despite strong directional selection, the tendency for a disproportionate large role of the X (Z) chromosome in post-zygotic isolation, and the evolution of sex.     

Estimation of DNA Sequence Context-dependent Mutation Rates Using Primate Genomic Sequences

It is understood that DNA and amino acid substitution rates are highly sequence context-dependent, e.g., C --> T substitutions in vertebrates may occur much more frequently at CpG sites and that cysteine substitution rates may depend on support of the context for participation in a disulfide bond. Furthermore, many applications rely on quantitative models of nucleotide or amino acid substitution, including phylogenetic inference and identification of amino acid sequence positions involved in functional specificity. We describe quantification of the context dependence of nucleotide substitution rates using baboon, chimpanzee, and human genomic sequence data generated by the NISC Comparative Sequencing Program. Relative mutation rates are reported for the 96 classes of mutations of the form 5' alphabetagamma 3' --> 5' alphadeltagamma 3', where alpha, beta, gamma, and delta are nucleotides and beta not equal delta, based on maximum likelihood calculations. Our results confirm that C --> T substitutions are enhanced at CpG sites compared with other transitions, relatively independent of the identity of the preceding nucleotide. While, as expected, transitions generally occur more frequently than transversions, we find that the most frequent transversions involve the C at CpG sites (CpG transversions) and that their rate is comparable to the rate of transitions at non-CpG sites. A four-class model of the rates of context-dependent evolution of primate DNA sequences, CpG transitions > non-CpG transitions approximately CpG transversions > non-CpG transversions, captures qualitative features of the mutation spectrum. We find that despite qualitative similarity of mutation rates among different genomic regions, there are statistically significant differences.     

Methylation levels at selected CpG sites in the factor VIII and FGFR3 genes, in mature female and male germ cells: implications for male-driven evolution.

Transitional mutations at CpG dinucleotides account for approximately a third of all point mutations. These mutations probably arise through spontaneous deamination of 5-methylcytosine. Studies of CpG mutation rates in disease-linked genes, such as factor VIII and FGFR3, have indicated that they more frequently originate in male than in female germ cells. It has been speculated that these sex-biased mutation rates might be a consequence of sex-specific methylation differences between the female and the male germ lines. Using the bisulfite-based genomic-sequencing method, we investigated the methylation status of the human factor VIII and FGFR3 genes in mature male and female germ cells. With the exception of a single CpG, both genes were found to be equally and highly methylated in oocytes and spermatocytes. Whereas these observations strongly support the notion that DNA methylation is the major determining factor for recurrent CpG germ-line mutations in patients with hemophilia and achondroplasia, the higher mutation rate in the male germ line is apparently not a simple reflection of sex-specific methylation differences.     

Germ-line origins of mutation in families with hemophilia B: the sex ratio varies with the type of mutation.

Previous epidemiological and biochemical studies have generated conflicting estimates of the sex ratio of mutation. Direct genomic sequencing in combination with haplotype analysis extends previous analyses by allowing the precise mutation to be determined in a given family. From analysis of the factor IX gene of 260 consecutive families with hemophilia B, we report the germ-line origin of mutation in 25 families. When combined with 14 origins of mutation reported by others and with 4 origins previously reported by us, a total of 25 occur in the female germ line, and 18 occur in the male germ line. The excess of germ-line origins in females does not imply an overall excess mutation rate per base pair in the female germ line. Bayesian analysis of the data indicates that the sex ratio varies with the type of mutation. The aggregate of single-base substitutions shows a male predominance of germ-line mutations (P < .002). The maximum-likelihood estimate of the male predominance is 3.5-fold. Of the single-base substitutions, transitions at the dinucleotide CpG show the largest male predominance (11-fold). In contrast to single-base substitutions, deletions display a sex ratio of unity. Analysis of the parental age at transmission of a new mutation suggests that germ-line mutations are associated with a small increase in parental age in females but little, if any, increase in males. Although direct genomic sequencing offers a general method for defining the origin of mutation in specific families, accurate estimates of the sex ratios of different mutational classes require large sample sizes and careful correction for multiple biases of ascertainment. The biases in the present data result in an underestimate of the enhancement of mutation in males.     

Accumulation and loss of asymmetric non-CpG methylation during male germ-cell development

DNA methylation is a well-characterized epigenetic modification involved in gene regulation and transposon silencing in mammals. It mainly occurs on cytosines at CpG sites but methylation at non-CpG sites is frequently observed in embryonic stem cells, induced pluriotent stem cells, oocytes and the brain. The biological significance of non-CpG methylation is unknown. Here, we show that non-CpG methylation is also present in male germ cells, within and around B1 retrotransposon sequences interspersed in the mouse genome. It accumulates in mitotically arrested fetal prospermatogonia and reaches the highest level by birth in a Dnmt3l-dependent manner. The preferential site of non-CpG methylation is CpA, especially CpApG and CpApC. Although CpApG (and CpTpG) sites contain cytosines at symmetrical positions, hairpin-bisulfite sequencing reveals that they are hemimethylated, suggesting the absence of a template-dependent copying mechanism. Indeed, the level of non-CpG methylation decreases after the resumption of mitosis in the neonatal period, whereas that of CpG methylation does not. The cells eventually lose non-CpG methylation by the time they become spermatogonia. Our results show that non-CpG methylation accumulates in non-replicating, arrested cells but is not maintained in mitotically dividing cells during male germ-cell development.     

Alu element mutation spectra: molecular clocks and the effect of DNA methylation

In primate genomes more than 40% of CpG islands are found within repetitive elements. With more than one million copies in the human genome, the Alu family of retrotransposons represents the most successful short interspersed element (SINE) in primates and CpG dinucleotides make up about 20% of Alu sequences. It is generally thought that CpG dinucleotides mutate approximately ten times faster than other dinucleotides due to cytosine methylation and the subsequent deamination and conversion of C-->T. However, the disparity of Alu subfamily age estimations based upon CpG or non-CpG substitution density indicates a more complex relationship between CpG and non-CpG substitutions within the Alu elements. Here we report an analysis of the mutation patterns for 5296 Alu elements comprising 20 subfamilies. Our results indicate a relatively constant CpG versus non-CpG substitution ratio of approximately 6 for the young (AluY) and intermediate (AluS) Alu subfamilies. However, a more complex non-linear relationship between CpG and non-CpG substitutions was observed when old (AluJ) subfamilies were included in the analysis. These patterns may be the result of the slowdown of the neutral mutation rate during primate evolution and/or an increase in the CpG mutation rate as the consequence of increased DNA methylation in response to a burst of retrotransposition activity approximately 35 million years ago.     

Local similarity in evolutionary rates extends over whole chromosomes in human-rodent and mouse-rat comparisons: implications for understanding the mechanistic basis of the male mutation bias

The sex chromosomes and autosomes spend different times in the germ line of the two sexes. If cell division is mutagenic and if the sexes differ in number of cell divisions, then we expect that sequences on the X and Y chromosomes and autosomes should mutate at different rates. Tests of this hypothesis for several mammalian species have led to conflicting results. At the same time, recent evidence suggests that the chromosomal location of genes on autosomes affects their rate of evolution at synonymous sites. This suggests a mutagenic source different from germ cell replication. To correctly interpret the previous estimates of male mutation bias, it is crucial to understand the degree and range of this local similarity. With a carefully chosen randomization protocol, local similarity in synonymous rates of evolution can be detected in human-rodent and mouse-rat comparisons. However, the synonymous-site similarity in the mouse-rat comparison remains weak. Simulations suggest that this difference between the mouse-human and the mouse-rat comparisons is not artifactual and that there is therefore a difference between humans and rodents in the local patterns of mutation or selection on synonymous sites (conversely, we show that the previously reported absence of a local similarity in nonsynonymous rates of evolution in the human-rodent comparison was a methodological artifact). We show that linkage effects have a long-range component: not one in a million random genomes shows such levels of autosomal heterogeneity. The heterogeneity is so great that more autosomes than expected by chance have rates of synonymous evolution comparable with that of the X chromosome. As autosomal heterogeneity cannot be owing to different times spent in the germ line, this demonstrates that the dominant determiner of synonymous rates of evolution is not, as has been conjectured, the time spent in the male germ line.     

Rates of DNA sequence evolution are not sex-biased in Drosophila melanogaster and D. simulans

To determine whether male- or female-biased mutation rates have affected the molecular evolution of Drosophila melanogaster and D. simulans, we calculated the male-to-female ratio of germline cell divisions ([symbol: see text]) from germline generation data and the male-to-female ratio of mutation rate ([symbol: see text]) by comparing chromosomal levels of nucleotide divergence. We found that the ratio of germline cell divisions changes from indicating a weak female bias to indicating a weak male bias as the age of reproduction increases. The range of [symbol: see text] values that we observed, however, does not lead us to expect much, if any, difference in mutation rate between the sexes. Silent and intron nucleotide divergence were compared between nine loci on the X chromosome and nine loci on the second and third chromosomes. The average levels of nucleotide divergence were not significantly different across the chromosomes, although both silent and intron sites show a trend toward slightly more divergence on the X. These results indicate a lack of sex- or chromosome-biased molecular evolution in D. melanogaster and D. simulans.      

Sperm competition can drive a male-biased mutation rate

A pattern of male-biased mutation has been found in a wide range of species. The standard explanation for this bias is that there are greater numbers of mitotic cell divisions in the history of the average sperm, compared to the average egg, and that mutations typically result from errors made during replication. However, this fails to provide an ultimate evolutionary explanation for why the male germline would tolerate more mutations that are typically deleterious. One possibility is that if there is a tradeoff between producing large numbers of sperm and expending energetic resources in maintaining a lower mutation rate, sperm competition would select for males that produce larger numbers of sperm despite a higher resulting mutation rate. Here I describe a model that jointly considers the fitness consequences of deleterious mutation and mating success in the face of sperm competition. I show that a moderate level of sperm competition can account for the observation that the male germline tolerates a higher mutation rate than the female germline.     

Maternal effect for DNA mismatch repair in the mouse.

DNA mismatch repair (DMR) functions to maintain genome stability. Prokaryotic and eukaryotic cells deficient in DMR show a microsatellite instability (MSI) phenotype characterized by repeat length alterations at microsatellite sequences. Mice deficient in Pms2, a mammalian homolog of bacterial mutL, develop cancer and display MSI in all tissues examined, including the male germ line where a frequency of approximately 10% was observed. To determine the consequences of maternal DMR deficiency on genetic stability, we analyzed F(1) progeny from Pms2(-/-) female mice mated with wild-type males. Our analysis indicates that MSI in the female germ line was approximately 9%. MSI was also observed in paternal alleles, a surprising result since the alleles were obtained from wild-type males and the embryos were therefore DMR proficient. We propose that mosaicism for paternal alleles is a maternal effect that results from Pms2 deficiency during the early cleavage divisions. The absence of DMR in one-cell embryos leads to the formation of unrepaired replication errors in early cell divisions of the zygote. The occurrence of postzygotic mutation in the early mouse embryo suggests that Pms2 deficiency is a maternal effect, one of a limited number identified in the mouse and the first to involve a DNA repair gene.     

Substitution rate variation at human CpG sites correlates with non-CpG divergence,methylation level and GC content

Background  

A major goal in the study of molecular evolution is to unravel the mechanisms that induce variation in the germ line mutation rate and in the genome-wide mutation profile. The rate of germ line mutation is considerably higher for cytosines at CpG sites than for any other nucleotide in the human genome, an increase commonly attributed to cytosine methylation at CpG sites. The CpG mutation rate, however, is not uniform across the genome and, as methylation levels have recently been shown to vary throughout the genome, it has been hypothesized that methylation status may govern variation in the rate of CpG mutation.     

Analysis of mutation rates in the SMCY/SMCX genes shows that mammalian evolution is male driven

Mammalian evolution is believed to be male driven because the greater number of germ cell divisions per generation in males increases the opportunity for errors in DNA replication. Since the Y Chromosome (Chr) replicates exclusively in males, its genes should also evolve faster than X or autosomal genes. In addition, estimating the overall male-to-female mutation ratio (α_m) is of great importance as a large α_m implies that replication-independent mutagenic events play a relatively small role in evolution. A small α_m suggests that the impact of these factors may, in fact, be significant. In order to address this problem, we have analyzed the rates of evolution in the homologous X-Y common SMCX/SMCY genes from three different species—mouse, human, and horse. The SMC genes were chosen because the X and Y copies are highly homologous, well conserved in evolution, and in all probability functionally interchangeable. Sequence comparisons and analysis of synonymous substitutions in approximately 1kb of the 5′ coding region of the SMC genes reveal that the Y-linked copies are evolving approximately 1.8 times faster than their X homologs. The male-to-female mutation ratio α_m was estimated to be 3. These data support the hypothesis that mammalian evolution is male driven. However, the ratio value is far smaller than suggested in earlier works, implying significance of replication-independent mutagenic events in evolution. Received: 18 April 1996 / Accepted: 4 October 1996     

Heterogeneous genomic molecular clocks in primates

Using data from primates, we show that molecular clocks in sites that have been part of a CpG dinucleotide in recent past (CpG sites) and non-CpG sites are of markedly different nature, reflecting differences in their molecular origins. Notably, single nucleotide substitutions at non-CpG sites show clear generation-time dependency, indicating that most of these substitutions occur by errors during DNA replication. On the other hand, substitutions at CpG sites occur relatively constantly over time, as expected from their primary origin due to methylation. Therefore, molecular clocks are heterogeneous even within a genome. Furthermore, we propose that varying frequencies of CpG dinucleotides in different genomic regions may have contributed significantly to conflicting earlier results on rate constancy of mammalian molecular clock. Our conclusion that different regions of genomes follow different molecular clocks should be considered when inferring divergence times using molecular data and in phylogenetic analysis.     

Assessing the underlying pattern of human germline mutations: lessons from the factor IX gene.

Germline mutations cause or predispose to most disease. Hemophilia B is a useful model for studying the underlying pattern of recent germline mutations in humans because the observed pattern of mutation in factor IX more closely reflects the underlying pattern of mutation than the observed pattern for many other genes. In addition, it is possible to identify and correct for biases inherent in ascertaining only those mutations that cause hemophilia. Aspects of the pattern of germline mutation in the factor IX gene are becoming clear: 1) in the United States, two-thirds of mutations causing mild disease arose from three founders whereas almost all the mutations resulting in either moderate or severe disease arose independently, generally within the past 150 years; 2) direct estimates of the rates of mutation in humans indicate that transitions are more frequent than transversions, which in turn are more frequent than deletions and insertions; 3) transitions at CpG are elevated approximately 24-fold relative to transitions at non-CpG dinucleotides; 4) transversions at CpG are elevated approximately eightfold relative to transversions at non-CpG dinucleotides; 5) the sum total of the dinucleotide mutation rates produces a bias against G and C bases that would be sufficient to maintain the G+C content of the factor IX gene at its evolutionarily conserved level of 40%; and 6) the pattern of mutation is similar for Caucasians residing in the United States and for Asians residing in Asia. Two ideas emerge from this and from an analysis of the pattern of recent deleterious mutations compared with ancient neutral mutations that have been fixed during evolution into the factor IX gene. First, the bulk of germline mutations are likely to arise from endogenous processes rather than environmental mutagens. Second, the factor IX protein is composed mostly of two classes of amino acids: critical residues in which all single-base missense changes will disrupt protein function, and "spacer" residues in which the precise nature of the residue is unimportant but the peptide bond is necessary to keep the critical residues in register. More work is necessary to assess the veracity and generality of these ideas.     

Effect of the assignment of ancestral CpG state on the estimation of nucleotide substitution rates in mammals

Background  

Molecular evolutionary studies in mammals often estimate nucleotide substitution rates within and outside CpG dinucleotides separately. Frequently, in alignments of two sequences, the division of sites into CpG and non-CpG classes is based simply on the presence or absence of a CpG dinucleotide in either sequence, a procedure that we refer to as CpG/non-CpG assignment. Although it likely that this procedure is biased, it is generally assumed that the bias is negligible if species are very closely related.     

Transmitted mutational events induced in mouse germ cells following acrylamide or glycidamide exposure

An increase in the germ line mutation rate in humans will result in an increase in the incidence of genetically determined diseases in subsequent generations. Thus, it is important to identify those agents that are mutagenic in mammalian germ cells. Acrylamide is water soluble, absorbed and distributed in the body, chemically reactive with nucleophilic sites, and there are known sources of human exposure. Here we review all seven published studies that assessed the effectiveness of acrylamide or its active metabolite, glycidamide, in inducing transmitted reciprocal translocations or gene mutations in the mouse. Major conclusions were (a) acrylamide is mutagenic in spermatozoa and spermatid stages of the male germ line; (b) in these spermatogenic stages acrylamide is mainly or exclusively a clastogen; (c) per unit dose, i.p. exposure is more effective than dermal exposure; and (d) per unit dose, glycidamide is more effective than acrylamide. Since stem cell spermatogonia persist and may accumulate mutations throughout the reproductive life of males, assessment of induced mutations in this germ cell stage is critical for the assessment of genetic risk associated with exposure to a mutagen. The two specific-locus mutation experiments which studied the stem cell spermatogonial stage yielded conflicting results. This discrepancy should be resolved. Finally, it is noted that no experiments have studied the mutagenic potential of acrylamide to increase the frequency of transmitted mutational events following exposure in the female germ line.     

Transmitted mutational events induced in mouse germ cells following acrylamide or glycidamide exposure

An increase in the germ line mutation rate in humans will result in an increase in the incidence of genetically determined diseases in subsequent generations. Thus, it is important to identify those agents that are mutagenic in mammalian germ cells. Acrylamide is water soluble, absorbed and distributed in the body, chemically reactive with nucleophilic sites, and there are known sources of human exposure. Here we review all seven published studies that assessed the effectiveness of acrylamide or its active metabolite, glycidamide, in inducing transmitted reciprocal translocations or gene mutations in the mouse. Major conclusions were (a) acrylamide is mutagenic in spermatozoa and spermatid stages of the male germ line; (b) in these spermatogenic stages acrylamide is mainly or exclusively a clastogen; (c) per unit dose, i.p. exposure is more effective than dermal exposure; and (d) per unit dose, glycidamide is more effective than acrylamide. Since stem cell spermatogonia persist and may accumulate mutations throughout the reproductive life of males, assessment of induced mutations in this germ cell stage is critical for the assessment of genetic risk associated with exposure to a mutagen. The two specific-locus mutation experiments which studied the stem cell spermatogonial stage yielded conflicting results. This discrepancy should be resolved. Finally, it is noted that no experiments have studied the mutagenic potential of acrylamide to increase the frequency of transmitted mutational events following exposure in the female germ line.