Mutational Dynamics of Microsatellites

Microsatellites are a ubiquitous class of simple repetitive DNA sequences, which are widespread in both eukaryotic and prokaryotic genomes. The use of microsatellites as polymorphic DNA markers has considerably increased both in the number of studies and in the number of organisms, primarily for genetic mapping, studying genomic instability in cancer, population genetics, forensics, conservation biology, molecular anthropology and in the studies of human evolutionary history. Although simple sequence repeats have been extensively used in studies encompassing varied areas of genetics, the mutation dynamics of these genome regions is still not well understood. The present review focuses on the mutational dynamics of microsatellite DNA with special reference to mutational mechanisms and their role in microsatellite evolution.     

Microsatellites: genomic distribution,putative functions and mutational mechanisms: a review

Microsatellites, or tandem simple sequence repeats (SSR), are abundant across genomes and show high levels of polymorphism. SSR genetic and evolutionary mechanisms remain controversial. Here we attempt to summarize the available data related to SSR distribution in coding and noncoding regions of genomes and SSR functional importance. Numerous lines of evidence demonstrate that SSR genomic distribution is nonrandom. Random expansions or contractions appear to be selected against for at least part of SSR loci, presumably because of their effect on chromatin organization, regulation of gene activity, recombination, DNA replication, cell cycle, mismatch repair system, etc. This review also discusses the role of two putative mutational mechanisms, replication slippage and recombination, and their interaction in SSR variation.     

CpG/CpNpG motifs in the coding region are preferred sites for mutagenesis in the breast cancer susceptibility genes

The range of BRCA1/BRCA2 gene mutations is diverse and the mechanism accounting for this heterogeneity is obscure. To gain insight into the endogenous mutational mechanisms involved, we evaluated the association of specific sequences (i.e. CpG/CpNpG motifs, homonucleotides, short repeats) and mutations within the genes. We classified 1337 published mutations in BRCA1 (1765 BRCA2 mutations) for each specific sequence, and employed computer simulation combined with mathematical calculations to estimate the true underlying tendency of mutation occurrence. Interestingly, we found no mutational bias to homonucleotides and repeats in deletions/insertions and substitutions but striking bias to CpG/CpNpG in substitutions in both genes. This suggests that methylation-dependent DNA alterations would be a major mechanism for mutagenesis.     

A role for selection in regulating the evolutionary emergence of disease-causing and other coding CAG repeats in humans and mice

The evolutionary expansion of CAG repeats in human triplet expansion disease genes is intriguing because of their deleterious phenotype. In the past, this expansion has been suggested to reflect a broad genomewide expansion of repeats, which would imply that mutational and evolutionary processes acting on repeats differ between species. Here, we tested this hypothesis by analyzing repeat- and flanking-sequence evolution in 28 repeat-containing genes that had been sequenced in humans and mice and by considering overall lengths and distributions of CAG repeats in the two species. We found no evidence that these repeats were longer in humans than in mice. We also found no evidence for preferential accumulation of CAG repeats in the human genome relative to mice from an analysis of the lengths of repeats identified in sequence databases. We then investigated whether sequence properties, such as base and amino acid composition and base substitution rates, showed any relationship to repeat evolution. We found that repeat-containing genes were enriched in certain amino acids, presumably as the result of selection, but that this did not reflect underlying biases in base composition. We also found that regions near repeats showed higher nonsynonymous substitution rates than the remainder of the gene and lower nonsynonymous rates in genes that contained a repeat in both the human and the mouse. Higher rates of nonsynonymous mutation in the neighborhood of repeats presumably reflect weaker purifying selection acting in these regions of the proteins, while the very low rate of nonsynonymous mutation in proteins containing a CAG repeat in both species presumably reflects a high level of purifying selection. Based on these observations, we propose that the mutational processes giving rise to polyglutamine repeats in human and murine proteins do not differ. Instead, we propose that the evolution of polyglutamine repeats in proteins results from an interplay between mutational processes and selection.     

Evolution of simple sequence repeat-mediated phase variation in bacterial genomes

Mutability as mechanism for rapid adaptation to environmental challenge is an alluringly simple concept whose apotheosis is realized in simple sequence repeats (SSR). Bacterial genomes of several species contain SSRs with a proven role in adaptation to environmental fluctuations. SSRs are hypermutable and generate reversible mutations in localized regions of bacterial genomes, leading to phase variable ON/OFF switches in gene expression. The application of genetic, bioinformatic, and mathematical/computational modeling approaches are revolutionizing our current understanding of how genomic molecular forces and environmental factors influence SSR-mediated adaptation and led to evolution of this mechanism of localized hypermutation in bacterial genomes.     

Molecular mechanisms of cardiomyogenesis and perspectives of cardiomyocyte regeneration in cardiac failure

This review analyzes the current state in investigations of molecular, genetic and cellular mechanisms of cardiac development as well as perspectives to use this knowledge for treatment of cardiac failure by means of replenishing cardiomyocytes in damaged myocardium.     

A novel mutational hotspot in a natural quasipalindrome in Escherichia coli

We have found that most spontaneous mutations in the thyA gene of Escherichia coli selected for resistance to trimethoprim result from a TA to AT transversion at a single site within an imperfect inverted repeat or quasipalindrome sequence. This natural quasipalindrome within the coding region of thyA contains an extraordinarily potent hotspot for mutation. Our analysis provides evidence that these mutations are templated by nearby sequences by replication within a hairpin structure. Although quasipalindrome-associated mutations have been observed in many organisms, including humans, the cellular avoidance mechanisms for these unusual mutational events have remained unexplored. We find that the mutational hotspot in thyA is dramatically stimulated by inactivation of exonucleases I and VII, which degrade single-strand DNA with a common 3'-5' polarity. We propose that these exonucleases abort the replicative misalignment events that initiate hairpin-templated mutagenesis by degrading displaced nascent DNA strands. Mismatch repair-defective strains also showed increased mutability at the hotspot, consistent with the notion that these mutations arise during chromosomal lagging-strand replication and are often subsequently removed by methyl-directed mismatch repair. The absence of the thyA quasipalindrome sequence from other related bacterial genera suggests that this sequence represents a "selfish" DNA element whose existence itself is driven by this unusual hairpin-templating mechanism.     

Theoretical analysis of mutation hotspots and their DNA sequence context specificity

Mutation frequencies vary significantly along nucleotide sequences such that mutations often concentrate at certain positions called hotspots. Mutation hotspots in DNA reflect intrinsic properties of the mutation process, such as sequence specificity, that manifests itself at the level of interaction between mutagens, DNA, and the action of the repair and replication machineries. The hotspots might also reflect structural and functional features of the respective DNA sequences. When mutations in a gene are identified using a particular experimental system, resulting hotspots could reflect the properties of the gene product and the mutant selection scheme. Analysis of the nucleotide sequence context of hotspots can provide information on the molecular mechanisms of mutagenesis. However, the determinants of mutation frequency and specificity are complex, and there are many analytical methods for their study. Here we review computational approaches for analyzing mutation spectra (distribution of mutations along the target genes) that include many mutable (detectable) positions. The following methods are reviewed: derivation of a consensus sequence, application of regression approaches to correlate nucleotide sequence features with mutation frequency, mutation hotspot prediction, analysis of oligonucleotide composition of regions containing mutations, pairwise comparison of mutation spectra, analysis of multiple spectra, and analysis of "context-free" characteristics. The advantages and pitfalls of these methods are discussed and illustrated by examples from the literature. The most reliable analyses were obtained when several methods were combined and information from theoretical analysis and experimental observations was considered simultaneously. Simple, robust approaches should be used with small samples of mutations, whereas combinations of simple and complex approaches may be required for large samples. We discuss several well-documented studies where analysis of mutation spectra has substantially contributed to the current understanding of molecular mechanisms of mutagenesis. The nucleotide sequence context of mutational hotspots is a fingerprint of interactions between DNA and DNA repair, replication, and modification enzymes, and the analysis of hotspot context provides evidence of such interactions.     

Mutation patterns of amino acid tandem repeats in the human proteome

Background

Amino acid tandem repeats are found in nearly one-fifth of human proteins. Abnormal expansion of these regions is associated with several human disorders. To gain further insight into the mutational mechanisms that operate in this type of sequence, we have analyzed a large number of mutation variants derived from human expressed sequence tags (ESTs).

Results

We identified 137 polymorphic variants in 115 different amino acid tandem repeats. Of these, 77 contained amino acid substitutions and 60 contained gaps (expansions or contractions of the repeat unit). The analysis showed that at least about 21% of the repeats might be polymorphic in humans. We compared the mutations found in different types of amino acid repeats and in adjacent regions. Overall, repeats showed a five-fold increase in the number of gap mutations compared to adjacent regions, reflecting the action of slippage within the repetitive structures. Gap and substitution mutations were very differently distributed between different amino acid repeat types. Among repeats containing gap variants we identified several disease and candidate disease genes.

Conclusion

This is the first report at a genome-wide scale of the types of mutations occurring in the amino acid repeat component of the human proteome. We show that the mutational dynamics of different amino acid repeat types are very diverse. We provide a list of loci with highly variable repeat structures, some of which may be potentially involved in disease.     

Advances in the application of germline tandem repeat instability for in situ monitoring

Alterations in tandem repetitive DNA sequences such as minisatellite DNA and expanded simple tandem repeats (ESTRs) may provide useful biomarkers of induced germline effects. In this review, I describe the differences between ESTRs and minisatellites with respect to their structure and mutational mechanisms, and discuss field applications measuring induced germline instability. It is evident that both types of loci have high rates of mutation that facilitate the measurement of induced mutation measured in relatively small numbers of samples following environmentally relevant exposures. Several research groups have used these loci to demonstrate a significant increase in germline mutation in humans and animals exposed to radioactive or chemical pollutants in their natural environment. Mutations are manifested as gains or losses in repeat units and are detected either by pedigree screening or by PCR amplification of sperm DNA. Mutations at both ESTRs and minisatellites appear to arise via indirect mechanisms rather than by direct damage to the repeat locus itself. Most interestingly, ESTR instability following radiation has been shown to be heritable and transmitted to subsequent generations. An understanding of the mechanisms involved in induced instability is required in order to begin to decipher the potential biological implications of increased germline tandem repeat mutation. Furthermore, relatively few studies have investigated the ability of different genotoxins to induce tandem repeat instability. Such laboratory-based experiments will be crucial in clarifying the particular environmental or occupational exposures that should be targeted for future studies and for isolating and subsequently identifying the putative mutagens in complex environmental matrices.     

Insights into mutagenesis using Escherichia coli chromosomal lacZ strains that enable detection of a wide spectrum of mutational events

Strand misalignments at DNA repeats during replication are implicated in mutational hotspots. To study these events, we have generated strains carrying mutations in the Escherichia coli chromosomal lacZ gene that revert via deletion of a short duplicated sequence or by template switching within imperfect inverted repeat (quasipalindrome, QP) sequences. Using these strains, we demonstrate that mutation of the distal repeat of a quasipalindrome, with respect to replication fork movement, is about 10-fold higher than the proximal repeat, consistent with more common template switching on the leading strand. The leading strand bias was lost in the absence of exonucleases I and VII, suggesting that it results from more efficient suppression of template switching by 3' exonucleases targeted to the lagging strand. The loss of 3' exonucleases has no effect on strand misalignment at direct repeats to produce deletion. To compare these events to other mutations, we have reengineered reporters (designed by Cupples and Miller 1989) that detect specific base substitutions or frameshifts in lacZ with the reverting lacZ locus on the chromosome rather than an F' element. This set allows rapid screening of potential mutagens, environmental conditions, or genetic loci for effects on a broad set of mutational events. We found that hydroxyurea (HU), which depletes dNTP pools, slightly elevated templated mutations at inverted repeats but had no effect on deletions, simple frameshifts, or base substitutions. Mutations in nucleotide diphosphate kinase, ndk, significantly elevated simple mutations but had little effect on the templated class. Zebularine, a cytosine analog, elevated all classes.     

核酸主动式杂交微电子芯片及其应用研究进展

介绍了基于电场作用下核酸主动杂交原理的微电子芯片的微加工方法、分析系统组成、杂交分析技术原理,综述了该技术在点突变分析、单核苷酸多态性分析、简单串联重复序列分析、芯片上多重链替代扩增、病原物检测等方面的应用进展。     

Bayesian analysis of mutational spectra

Studies that examine both the frequency of gene mutation and the pattern or spectrum of mutational changes can be used to identify chemical mutagens and to explore the molecular mechanisms of mutagenesis. In this article, we propose a Bayesian hierarchical modeling approach for the analysis of mutational spectra. We assume that the total number of independent mutations and the numbers of mutations falling into different response categories, defined by location within a gene and/or type of alteration, follow binomial and multinomial sampling distributions, respectively. We use prior distributions to summarize past information about the overall mutation frequency and the probabilities corresponding to the different mutational categories. These priors can be chosen on the basis of data from previous studies using an approach that accounts for heterogeneity among studies. Inferences about the overall mutation frequency, the proportions of mutations in each response category, and the category-specific mutation frequencies can be based on posterior distributions, which incorporate past and current data on the mutant frequency and on DNA sequence alterations. Methods are described for comparing groups and for assessing dose-related trends. We illustrate our approach using data from the literature.     

Sequence-dependent effect of interruptions on microsatellite mutation rate in mismatch repair-deficient human cells

Although microsatellite mutation rates generally increase with increasing length of the repeat tract, interruptions in a microsatellite may stabilize it. We have performed a direct analysis of the effect of microsatellite interruptions on mutation rate and spectrum in cultured mammalian cells. Two mononucleotide sequences (G₁₇ and A₁₇) and a dinucleotide [(CA)₁₇] were compared with interrupted repeats of the same size and with sequences of 8 repeat units. MMR-deficient (MMR⁻) cells were used for these studies to eliminate effects of this repair process. Mutation rates were determined by fluctuation analysis on cells containing a microsatellite sequence at the 5′ end of an antibiotic-resistance gene; the vector carrying this sequence was integrated in the genome of the cells. In general, interrupted sequences had lower mutation rates than perfect ones of the same size, but the magnitude of the difference was dependent upon the sequence of the interrupting base(s). Some interrupted repeats had mutation rates that were lower than those of perfect sequences of the same length but similar to those of half the length. This suggests that interrupting bases effectively divide microsatellites into smaller repeat runs with mutational characteristics different from those of the corresponding full-length microsatellite. We conclude that interruptions decrease microsatellite mutation rate and influence the spectrum of frameshift mutations. The sequence of the interrupting base(s) determines the magnitude of the effect on mutation rate.     

Perspectives of genomic diversification and molecular recombination towards R-gene evolution in plants

Plants are under strong evolutionary pressure in developing new and noble R genes to recognize pathogen avirulence (avr) determinants and bring about stable defense for generation after generations. Duplication, sequence variation by mutation, disparity in the length and structure of leucine rich repeats etc., causes tremendous variations within and among R genes in a plant thereby developing diverse recognitional specificity suitable enough for defense against new pathogens. Recent studies on genome sequencing, diversity and population genetics in different plants have thrown new insights on the molecular evolution of these genes. Tandem and segmental duplication are important factors in R gene abundance as inferred from the distribution of major nucleotide binding site-leucine rich repeats (NBS-LRRs) type R-genes in plant genomes. Likewise, R-gene evolution is also thought to be facilitated by cluster formation thereby causing recombination and sequence exchange and resulting in haplotypic diversity. Population studies have further proven that balancing selection is responsible for the maintenance of allelic diversity in R genes. In this review, we emphasize and discuss on improved perspectives towards the molecular mechanisms and selection pressure responsible for the evolution of NBS-LRR class resistance genes in plants.     

Evidence That Mutation Is Universally Biased towards AT in Bacteria

Mutation is the engine that drives evolution and adaptation forward in that it generates the variation on which natural selection acts. Mutation is a random process that nevertheless occurs according to certain biases. Elucidating mutational biases and the way they vary across species and within genomes is crucial to understanding evolution and adaptation. Here we demonstrate that clonal pathogens that evolve under severely relaxed selection are uniquely suitable for studying mutational biases in bacteria. We estimate mutational patterns using sequence datasets from five such clonal pathogens belonging to four diverse bacterial clades that span most of the range of genomic nucleotide content. We demonstrate that across different types of sites and in all four clades mutation is consistently biased towards AT. This is true even in clades that have high genomic GC content. In all studied cases the mutational bias towards AT is primarily due to the high rate of C/G to T/A transitions. These results suggest that bacterial mutational biases are far less variable than previously thought. They further demonstrate that variation in nucleotide content cannot stem entirely from variation in mutational biases and that natural selection and/or a natural selection-like process such as biased gene conversion strongly affect nucleotide content.     

Common deletion of SMAD4 in juvenile polyposis is a mutational hotspot

Juvenile polyposis (JP) is an autosomal dominant syndrome in which affected patients develop upper- and/or lower-gastrointestinal (GI) polyps. A subset of families with JP have germline mutations in the SMAD4 (MADH4) gene and are at increased risk of GI cancers. To date, six families with JP have been described as having the same SMAD4 deletion (1244-1247delAGAC). The objective of the present study is to determine whether this deletion is a common ancestral mutation or a mutational hotspot. DNA from members of four families with JP, from Iowa, Mississippi, Texas, and Finland, that had this 4-bp deletion was used to genotype 15 simple tandem repeat polymorphism (STRP) markers flanking the SMAD4 gene, including 2 new STRPs within 6.3 and 70.9 kb of the deletion. Haplotypes cosegregating with JP in each family were constructed, and the distances of the closest markers were determined from the draft sequence of the human genome. No common haplotype was observed in these four families with JP. A 14-bp region containing the deletion had four direct repeats and one inverted repeat. Because no common ancestor was suggested by haplotype analysis and the sequence flanking the deletion contains repeats frequently associated with microdeletions, this common SMAD4 deletion in JP most likely represents a mutational hotspot.     

Complex evolution of a salmonid microsatellite locus and its consequences in inferring allelic divergence from size information

Sequence analysis of 78 alleles of a microsatellite locus was resolved at various taxonomic levels among salmonid fishes to test for congruence between size and sequence information. Allelic variability of this locus involved changes in number of dinucleotide repeats as expected for microsatellite loci. However, additional mutational events involving large indels and base substitutions also occurred frequently among and within species. These caused the evolution of different imperfect microsatellite variants, from an ancestral perfect one, which was undetectable from allelic size information only. Alleles of the same length resulting from different mutational events were also observed among and within species. Altogether, these results revealed the incongruence between size and number of mutation events, implying that it may be imprecise to interpret mutational rates and relationships on the basis of size information alone in interspecific comparisons and, to a lesser extent, within species.