回文序列诱导突变的机制及人类相关疾病

在原核和真核生物基因组中, 含有回文序列的区域高度可变且稳定性差, 主要原因是回文序列能形成发卡或十字形二级结构, 然后通过滑动错配、单链复性以及非同源末端连接(Non-homologous end joining, NHEJ)等机制导致缺失突变或染色体易位的发生。在人类基因组中, 回文序列较普遍存在于基因表达调控的重要作用元件中, 它诱导的缺失和易位突变还与男性不育、地中海贫血等多种疾病的发生、发展密切相关。文章综合近几年国内外相关文献, 初步阐释回文序列诱导突变的类型和可能机制, 及其与人类疾病的关系, 为进一步探讨回文序列在基因表达调控、基因突变及人类疾病中的作用及功能等相关研究提供参考。     

Palindrome regeneration by template strand-switching mechanism at the origin of DNA replication of porcine circovirus via the rolling-circle melting-pot replication model

Palindromic sequences (inverted repeats) flanking the origin of DNA replication with the potential of forming single-stranded stem-loop cruciform structures have been reported to be essential for replication of the circular genomes of many prokaryotic and eukaryotic systems. In this study, mutant genomes of porcine circovirus with deletions in the origin-flanking palindrome and incapable of forming any cruciform structures invariably yielded progeny viruses containing longer and more stable palindromes. These results suggest that origin-flanking palindromes are essential for termination but not for initiation of DNA replication. Detection of template strand switching in the middle of an inverted repeat strand among the progeny viruses demonstrated that both the minus genome and a corresponding palindromic strand served as templates simultaneously during DNA biosynthesis and supports the recently proposed rolling-circle "melting-pot" replication model. The genome configuration presented by this model, a four-stranded tertiary structure, provides insights into the mechanisms of DNA replication, inverted repeat correction (or conversion), and illegitimate recombination of any circular DNA molecule with an origin-flanking palindrome.     

The relationship between palindrome avoidance and intragenic codon usage variations: a Monte Carlo study

Several studies have shown that codon usage within genes varies, as it seems dependent on both codon context and codon position within the gene. Given that palindromes in addition often are avoided in genomes, this study aimed at finding out if intragenic variations in codon usage may be a way to control the amount and location of palindromes. A Monte Carlo algorithm was written which resampled the codons in genes while keeping the amino acid sequence of the translation product constant. On the resampled sequences, palindromes were counted and their intragenic positions mapped. Escherichia coli K12 uses type II restriction-modification systems and displays pronounced codon usage phenomena. Using this as a reference organism it was clearly shown that the number of palindromes in genes is generally lower than the amount of palindromes in resampled genes; thus, the succession of codons seems to be a way to decrease the number of palindromes. The intragenic position of palindromes in resampled sequences, however, was largely equal to the position in the native genes, so codon usage phenomena are unlikely to be a way to control the intragenic position of palindromes. The analysis was repeated on two bacteriophages and gave similar same results, even though the virus genomes are much smaller. Studies on the endosymbionts Buchnera sp. APS and Wigglesworthia sp., which seemingly have no type II restriction-modification systems, showed that in these species there is only weak evidence for codon usage acting to control the number of palindromes.     

Species-specific Typing of DNA Based on Palindrome Frequency Patterns

DNA in its natural, double-stranded form may contain palindromes, sequences which read the same from either side because they are identical to their reverse complement on the sister strand. Short palindromes are underrepresented in all kinds of genomes. The frequency distribution of short palindromes exhibits more than twice the inter-species variance of non-palindromic sequences, which renders palindromes optimally suited for the typing of DNA. Here, we show that based on palindrome frequency, DNA sequences can be discriminated to the level of species of origin. By plotting the ratios of actual occurrence to expectancy, we generate palindrome frequency patterns that allow to cluster different sequences of the same genome and to assign plasmids, and in some cases even viruses to their respective host genomes. This finding will be of use in the growing field of metagenomics.     

Formation of large palindromic DNA by homologous recombination of short inverted repeat sequences in Saccharomyces cerevisiae

Large DNA palindromes form sporadically in many eukaryotic and prokaryotic genomes and are often associated with amplified genes. The presence of a short inverted repeat sequence near a DNA double-strand break has been implicated in the formation of large palindromes in a variety of organisms. Previously we have established that in Saccharomyces cerevisiae a linear DNA palindrome is efficiently formed from a single-copy circular plasmid when a DNA double-strand break is introduced next to a short inverted repeat sequence. In this study we address whether the linear palindromes form by an intermolecular reaction (that is, a reaction between two identical fragments in a head-to-head arrangement) or by an unusual intramolecular reaction, as it apparently does in other examples of palindrome formation. Our evidence supports a model in which palindromes are primarily formed by an intermolecular reaction involving homologous recombination of short inverted repeat sequences. We have also extended our investigation into the requirement for DNA double-strand break repair genes in palindrome formation. We have found that a deletion of the RAD52 gene significantly reduces palindrome formation by intermolecular recombination and that deletions of two other genes in the RAD52-epistasis group (RAD51 and MRE11) have little or no effect on palindrome formation. In addition, palindrome formation is dramatically reduced by a deletion of the nucleotide excision repair gene RAD1.     

Short interrupted palindromes on the extragenic DNA of Escherichia coli K-12, Haemophilus influenzae and Neisseria meningitidis

MOTIVATION: The importance of the various kinds of repetitive nucleotide sequences for the workings of bacterial DNA has been widely recognized. This work is concerned with the distribution of a particular group of repetitive sequences, the short-sequenced interrupted extragenic palindromes, on the genetic maps of Escherichia coli K-12, Haemophilus influenzae Rd and Neisseria meningitidis Z2491 and MC58. A tool has been developed based upon a statistical hypothesis test taking into account the markovian structure of random sequences in order to determine the non-random character of extragenic palindromes. RESULTS: Totals of 7631, 12904, 4722 and 5477 non-random short interrupted palindromes have been found on the E.coli, H.influenzae, and N.meningitidis serogroup A and serogroup B genomes, respectively. Their distribution patterns on the respective genomes vary according to the bacterial species considered. Based on their position on the genome, palindromes could be distinguished as those which integrate longer, repetitive sequences; those which stand in isolation, and still others are associated to specific genome sites. AVAILABILITY: The complete list of the observed palindromes is available at the site http://www/lncc.br/~atrv. CONTACT: atrv@lncc.br     

Assessing Diversity of DNA Structure-Related Sequence Features in Prokaryotic Genomes

Prokaryotic genomes are diverse in terms of their nucleotide and oligonucleotide composition as well as presence of various sequence features that can affect physical properties of the DNA molecule. We present a survey of local sequence patterns which have a potential to promote non-canonical DNA conformations (i.e. different from standard B-DNA double helix) and interpret the results in terms of relationships with organisms'' habitats, phylogenetic classifications, and other characteristics. Our present work differs from earlier similar surveys not only by investigating a wider range of sequence patterns in a large number of genomes but also by using a more realistic null model to assess significant deviations. Our results show that simple sequence repeats and Z-DNA-promoting patterns are generally suppressed in prokaryotic genomes, whereas palindromes and inverted repeats are over-represented. Representation of patterns that promote Z-DNA and intrinsic DNA curvature increases with increasing optimal growth temperature (OGT), and decreases with increasing oxygen requirement. Additionally, representations of close direct repeats, palindromes and inverted repeats exhibit clear negative trends with increasing OGT. The observed relationships with environmental characteristics, particularly OGT, suggest possible evolutionary scenarios of structural adaptation of DNA to particular environmental niches.     

Characterization of species-specific repeats in 613 prokaryotic species

Prokaryotes are in general believed to possess small, compactly organized genomes, with repetitive sequences forming only a small part of them. Nonetheless, many prokaryotic genomes in fact contain species-specific repeats (>85 bp long genomic sequences with less than 60% identity to other species) as we have previously demonstrated. However, it is not known at present how frequent such species-specific repeats are and what their functional roles in bacterial genomes may be. Therefore, we have conducted a comprehensive survey of prokaryotic species-specific repeats and characterized them to examine as to whether there are functional classes among different repeats or not and how they are mutually related to each other. Of the 613 distinct prokaryotic species analyzed, 97% were found to contain at least one species-specific repeats. It seems interesting to note that the species-specific repeats thus identified appear to be functionally variable in different genomes: in some genomes, they are mostly associated with duplicated protein-coding genes, whereas in some other genomes with rRNA and tRNA genes. Contrary to what may be expected, only one-fourth of the species-specific repeats were found to be associated with mobile genetic elements.     

Microbial species delineation using whole genome sequences

Increased sequencing of microbial genomes has revealed that prevailing prokaryotic species assignments can be inconsistent with whole genome information for a significant number of species. The long-standing need for a systematic and scalable species assignment technique can be met by the genome-wide Average Nucleotide Identity (gANI) metric, which is widely acknowledged as a robust measure of genomic relatedness. In this work, we demonstrate that the combination of gANI and the alignment fraction (AF) between two genomes accurately reflects their genomic relatedness. We introduce an efficient implementation of AF,gANI and discuss its successful application to 86.5M genome pairs between 13,151 prokaryotic genomes assigned to 3032 species. Subsequently, by comparing the genome clusters obtained from complete linkage clustering of these pairs to existing taxonomy, we observed that nearly 18% of all prokaryotic species suffer from anomalies in species definition. Our results can be used to explore central questions such as whether microorganisms form a continuum of genetic diversity or distinct species represented by distinct genetic signatures. We propose that this precise and objective AF,gANI-based species definition: the MiSI (Microbial Species Identifier) method, be used to address previous inconsistencies in species classification and as the primary guide for new taxonomic species assignment, supplemented by the traditional polyphasic approach, as required.     

Functional gene networks based on the gene neighborhood in metagenomes

The gene neighborhood in prokaryotic genomes has been effectively utilized in inferring co-functional networks in various organisms. Previously, such genomic context information has been sought among completely assembled prokaryotic genomes. Here, we present a method to infer functional gene networks according to the gene neighborhood in metagenome contigs, which are incompletely assembled genomic fragments. Given that the amount of metagenome sequence data has now surpassed that of completely assembled prokaryotic genomes in the public domain, we expect benefits of inferring networks by the metagenome-based gene neighborhood. We generated co-functional networks for diverse taxonomical species using metagenomics contigs derived from the human microbiome and the ocean microbiome. We found that the networks based on the metagenome gene neighborhood outperformed those based on 1748 completely assembled prokaryotic genomes. We also demonstrated that the metagenome-based gene neighborhood could predict genes related to virulence-associated phenotypes in a bacterial pathogen, indicating that metagenome-based functional links could be sufficiently predictive for some phenotypes of medical importance. Owing to the exponential growth of metagenome sequence data in public repositories, metagenome-based inference of co-functional networks will facilitate understanding of gene functions and pathways in diverse species.     

The Epigenomic Landscape of Prokaryotes

DNA methylation acts in concert with restriction enzymes to protect the integrity of prokaryotic genomes. Studies in a limited number of organisms suggest that methylation also contributes to prokaryotic genome regulation, but the prevalence and properties of such non-restriction-associated methylation systems remain poorly understood. Here, we used single molecule, real-time sequencing to map DNA modifications including m6A, m4C, and m5C across the genomes of 230 diverse bacterial and archaeal species. We observed DNA methylation in nearly all (93%) organisms examined, and identified a total of 834 distinct reproducibly methylated motifs. This data enabled annotation of the DNA binding specificities of 620 DNA Methyltransferases (MTases), doubling known specificities for previously hard to study Type I, IIG and III MTases, and revealing their extraordinary diversity. Strikingly, 48% of organisms harbor active Type II MTases with no apparent cognate restriction enzyme. These active ‘orphan’ MTases are present in diverse bacterial and archaeal phyla and show motif specificities and methylation patterns consistent with functions in gene regulation and DNA replication. Our results reveal the pervasive presence of DNA methylation throughout the prokaryotic kingdoms, as well as the diversity of sequence specificities and potential functions of DNA methylation systems.     

An unsupervised classification scheme for improving predictions of prokaryotic TIS

Background  

Although it is not difficult for state-of-the-art gene finders to identify coding regions in prokaryotic genomes, exact prediction of the corresponding translation initiation sites (TIS) is still a challenging problem. Recently a number of post-processing tools have been proposed for improving the annotation of prokaryotic TIS. However, inherent difficulties of these approaches arise from the considerable variation of TIS characteristics across different species. Therefore prior assumptions about the properties of prokaryotic gene starts may cause suboptimal predictions for newly sequenced genomes with TIS signals differing from those of well-investigated genomes.     

Analysis of the Relationship between Genomic GC Content and Patterns of Base Usage,Codon Usage and Amino Acid Usage in Prokaryotes: Similar GC Content Adopts Similar Compositional Frequencies Regardless of the Phylogenetic Lineages

The GC contents of 2670 prokaryotic genomes that belong to diverse phylogenetic lineages were analyzed in this paper. These genomes had GC contents that ranged from 13.5% to 74.9%. We analyzed the distance of base frequencies at the three codon positions, codon frequencies, and amino acid compositions across genomes with respect to the differences in the GC content of these prokaryotic species. We found that although the phylogenetic lineages were remote among some species, a similar genomic GC content forced them to adopt similar base usage patterns at the three codon positions, codon usage patterns, and amino acid usage patterns. Our work demonstrates that in prokaryotic genomes: a) base usage, codon usage, and amino acid usage change with GC content with a linear correlation; b) the distance of each usage has a linear correlation with the GC content difference; and c) GC content is more essential than phylogenetic lineage in determining base usage, codon usage, and amino acid usage. This work is exceptional in that we adopted intuitively graphic methods for all analyses, and we used these analyses to examine as many as 2670 prokaryotes. We hope that this work is helpful for understanding common features in the organization of microbial genomes.     

A Study on Mutational Dynamics of Simple Sequence Repeats in Relation to Mismatch Repair System in Prokaryotic Genomes

Mutational bias toward expansion or contraction of simple sequence repeats (SSRs) is referred to as directionality of SSR evolution. In this communication, we report the mutational bias exhibited by mononucleotide SSRs occurring in the non-coding regions of several prokaryotic genomes. Our investigations revealed that the strains or species lacking mismatch repair (MMR) system generally show higher number of polymorphic SSRs than those species/strains having MMR system. An exception to this observation was seen in the mycobacterial genomes that are MMR deficient where only a few SSR tracts were seen with mutations. This low incidence of SSR mutations even in the MMR-deficient background could be attributed to the high fidelity of the DNA polymerases as a consequence of high generation time of the mycobacteria. MMR system-deficient species generally did not show any bias toward mononucleotide SSR expansions or contractions indicating a neutral evolution of SSRs in these species. The MMR-proficient species in which the observed mutations correspond to secondary mutations showed bias toward contraction of polymononucleotide tracts, perhaps, indicating low efficiency of MMR system to repair SSR-induced slippage errors on template strands. This bias toward deletion in the mononucleotide SSR tracts might be a probable reason behind scarcity for long poly A|T and G|C tracts in prokaryotic systems which are mostly MMR proficient. In conclusion, our study clearly demonstrates mutational dynamics of SSRs in relation to the presence/absence of MMR system in the prokaryotic system.     

Microbial systematics in the post-genomics era

Microbial systematics and phylogeny should form the foundation and guiding light for a comprehensive understanding of different aspects of microbiology. However, there are many critical issues in microbial systematics that are currently not resolved. Some of these include: how to define and delimit a prokaryotic species; development of rationale criteria for the assignment of higher taxonomic ranks; understanding what unique properties distinguish species from different groups; and understanding the branching order and interrelationship among higher prokaryotic clades. The sequencing of genomes from large numbers of cultured as well as uncultured microbes covering prokaryotic diversity provides unique means to achieve these important objectives. Prokaryotic genomes are found to be very diverse and dynamic and horizontal gene transfers (HGTs) are indicated to have played important role in species/genome evolution. Although HGT adds a layer of complexity in terms of understanding the genomes and species evolution, it is contended that vast majority of genes and genetic characteristics that are distinctive characteristics of higher prokaryotic taxa are vertically inherited and based on them a solid foundation for microbial systematics can be developed. We describe two kinds of molecular markers consisting of conserved indels in protein sequences and whole proteins that are specific for different groups that are proving particularly valuable in defining different prokaryotic groups in clear molecular terms and in understanding their interrelationships. The genetic and biochemical studies on these taxa-specific molecular markers also open the way to discover novel biochemical and physiological characteristics that are unique properties of these groups.     

Compound microsatellites in complete Escherichia coli genomes

Compound microsatellites consisting of two or more repeats in close proximity have been found in eukaryotic genomes. So far such compound microsatellites have not been investigated in any prokaryotic genomes. We have therefore examined compound microsatellites in 22 complete genomes of Escherichia coli, which is one of the ideal model organisms to analyze the nature and evolution of prokaryotic compound microsatellites. Our results indicated that about 1.75-2.85% of all microsatellites could be accounted as compound microsatellites with very low complexity, and most compound microsatellites were composed of very different motifs. Compound microsatellites were significantly overrepresented in all surveyed genomes. These results were dramatically different from those in eukaryotes. We discussed the possible reasons for the observed divergence.     

A Novel Prokaryotic Promoter Identified in the Genome of Some Monopartite Begomoviruses

Geminiviruses are known to exhibit both prokaryotic and eukaryotic features in their genomes, with the ability to express their genes and even replicate in bacterial cells. We have demonstrated previously the existence of unit-length single-stranded circular DNAs of Ageratum yellow vein virus (AYVV, a species in the genus Begomovirus, family Geminiviridae) in Escherichia coli cells, which prompted our search for unknown prokaryotic functions in the begomovirus genomes. By using a promoter trapping strategy, we identified a novel prokaryotic promoter, designated AV3 promoter, in nts 762-831 of the AYVV genome. Activity assays revealed that the AV3 promoter is strong, unidirectional, and constitutive, with an endogenous downstream ribosome binding site and a translatable short open reading frame of eight amino acids. Sequence analyses suggested that the AV3 promoter might be a remnant of prokaryotic ancestors that could be related to certain promoters of bacteria from marine or freshwater environments. The discovery of the prokaryotic AV3 promoter provided further evidence for the prokaryotic origin in the evolutionary history of geminiviruses.     

GAP-Seq: a method for identification of DNA palindromes

Background

Closely spaced long inverted repeats, also known as DNA palindromes, can undergo intrastrand annealing to form DNA hairpins. The ability to form these hairpins results in genome instability, difficulties in maintaining clones in Escherichia coli and major problems for most DNA sequencing approaches. Because of their role in genomic instability and gene amplification in some human cancers, it is important to develop systematic approaches to detect and characterize DNA palindromes.

Results

We developed a new protocol to identify palindromes that couples the S1 nuclease treated Cot0 DNA (GAPF) with high-throughput sequencing (GAP-Seq). Unlike earlier protocols, it does not involve restriction enzymatic digestion prior to DNA snap-back thereby preserving longer DNA sequences. It also indicates the location of the novel junction, which can then be recovered. Using MCF-7 breast cancer cell line as the proof-of-principle analysis, we have identified 35 palindrome candidates and physically characterized the top 5 candidates and their junctions. Because this protocol eliminates many of the false positives that plague earlier techniques, we have improved palindrome identification.

Conclusions

The GAP-Seq approach underscores the importance of developing new tools for identifying and characterizing palindromes, and provides a new strategy to systematically assess palindromes in genomes. It will be useful for studying human cancers and other diseases associated with palindromes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-394) contains supplementary material, which is available to authorized users.