Triple-helix potential of the mouse genome

Certain DNA sequences, including mirror-symmetric polypyrimidine/polypurine runs, are capable of folding into a triple-helix-containing non-B-form DNA structure called H-DNA. Such H-DNA-forming sequences are frequent in many eukaryotic genomes, including in mammals, and multiple lines of evidence indicate that these motifs are mutagenic and can impinge on DNA replication, transcription, and other aspects of genome function. In this study, we show that the triplex-forming potential of H-DNA motifs in the mouse genome can be evaluated using S1-sequencing (S1-seq), which uses the single-stranded DNA (ssDNA)-specific nuclease S1 to generate deep-sequencing libraries that report on the position of ssDNA throughout the genome. When S1-seq was applied to genomic DNA isolated from mouse testis cells and splenic B cells, we observed prominent clusters of S1-seq reads that appeared to be independent of endogenous double-strand breaks, that coincided with H-DNA motifs, and that correlated strongly with the triplex-forming potential of the motifs. Fine-scale patterns of S1-seq reads, including a pronounced strand asymmetry in favor of centrally-positioned reads on the pyrimidine-containing strand, suggested that this S1-seq signal is specific for one of the four possible isomers of H-DNA (H-y5). By leveraging the abundance and complexity of naturally occurring H-DNA motifs across the mouse genome, we further defined how polypyrimidine repeat length and the presence of repeat-interrupting substitutions modify the structure of H-DNA. This study provides a new approach for studying DNA secondary structure genome wide at high spatial resolution.

The mtDNA mutation spectrum in the PolG mutator mouse reveals germline and somatic selection

Background Mitochondrial DNA (mtDNA) codes for products necessary for electron transport and mitochondrial gene translation. mtDNA mutations can lead to human disease and influence organismal fitness. The PolG mutator mouse lacks mtDNA proofreading function and rapidly accumulates mtDNA mutations, making it a model for examining the causes and consequences of mitochondrial mutations. Premature aging in PolG mice and their physiology have been examined in depth, but the location, frequency, and diversity of their mtDNA mutations remain understudied. Identifying the locations and spectra of mtDNA mutations in PolG mice can shed light on how selection shapes mtDNA, both within and across organisms. Results Here, we characterized somatic and germline mtDNA mutations in brain and liver tissue of PolG mice to quantify mutation count (number of unique mutations) and frequency (mutation prevalence). Overall, mtDNA mutation count and frequency were the lowest in the D-loop, where an mtDNA origin of replication is located, but otherwise uniform across the mitochondrial genome. Somatic mtDNA mutations have a higher mutation count than germline mutations. However, germline mutations maintain a higher frequency and were also more likely to be silent. Cytosine to thymine mutations characteristic of replication errors were the plurality of basepair changes, and missense C to T mutations primarily resulted in increased protein hydrophobicity. Unlike wild type mice, PolG mice do not appear to show strand asymmetry in mtDNA mutations. Indel mutations had a lower count and frequency than point mutations and tended to be short, frameshift deletions. Conclusions Our results provide strong evidence that purifying selection plays a major role in the mtDNA of PolG mice. Missense mutations were less likely to be passed down in the germline, and they were less likely to spread to high frequencies. The D-loop appears to have resistance to mutations, either through selection or as a by-product of replication processes. Missense mutations that decrease hydrophobicity also tend to be selected against, reflecting the membrane-bound nature of mtDNA-encoded proteins. The abundance of mutations from polymerase errors compared with reactive oxygen species (ROS) damage supports previous studies suggesting ROS plays a minimal role in exacerbating the PolG phenotype, but our findings on strand asymmetry provide discussion for the role of polymerase errors in wild type organisms. Our results provide further insight on how selection shapes mtDNA mutations and on the aging mechanisms in PolG mice.

MutationalPatterns: The one stop shop for the analysis of mutational processes

Background: The collective of somatic mutations in a genome represents a record of mutational processes that have been operative in a cell. These processes can be investigated by extracting relevant mutational patterns from sequencing data. Results: Here, we present the next version of MutationalPatterns, an R/Bioconductor package, which allows in-depth mutational analysis of catalogues of single and double base substitutions as well as small insertions and deletions. Major features of the package include the possibility to perform regional mutation spectra analyses and the possibility to detect strand asymmetry phenomena, such as lesion segregation. On top of this, the package also contains functions to determine how likely it is that a signature can cause damaging mutations (i.e., mutations that affect protein function). This updated package supports stricter signature refitting on known signatures in order to prevent overfitting. Using simulated mutation matrices containing varied signature contributions, we showed that reliable refitting can be achieved even when only 50 mutations are present per signature. Additionally, we incorporated bootstrapped signature refitting to assess the robustness of the signature analyses. Finally, we applied the package on genome mutation data of cell lines in which we deleted specific DNA repair processes and on large cancer datasets, to show how the package can be used to generate novel biological insights. Conclusions: This novel version of MutationalPatterns allows for more comprehensive analyses and visualization of mutational patterns in order to study the underlying processes. Ultimately, in-depth mutational analyses may contribute to improved biological insights in mechanisms of mutation accumulation as well as aid cancer diagnostics. MutationalPatterns is freely available at http://bioconductor.org/packages/MutationalPatterns.

Polymorphism at four-fold degenerate site, but not at the intergenic regions, explains nucleotide compositional strand asymmetry in bacteria

We investigated single nucleotide polymorphism in intergenic regions (IRs) and four-fold degenerate sites (FFS) in genomes of three γ-Proteobacteria and two Firmicutes to understand the mechanism of nucleotide compositional asymmetry between the leading and the lagging strands. Pattern of the polymorphism spectra were alike regarding transitions but variable regarding transversions in the IRs of these bacteria. Contrasting trends of complementary polymorphisms such as C->T vs G->A as well as A->G vs T->C in the IRs vindicated similar replication-associated strand asymmetry regarding cytosine and adenine deamination, respectively, across these bacteria. Surprisingly, the polymorphism pattern at FFS was different from that of the IRs and its frequency was always more than the IRs in these bacteria. Further, the polymorphism patterns within a bacterium were inconsistent across the five amino acids, which neither the replication nor the transcription-associated mutations could explain. However, the polymorphism at FFS coincided with amino acid specific codon usage bias in the five bacteria. Further, strand asymmetry in nucleotide composition could be explained by the polymorphism at FFS, not at the IRs. Therefore, polymorphisms at FFS might not be treated as nearly neutral unlike that in IRs in these bacteria.

Exons and introns exhibit transcriptional strand asymmetry of dinucleotide distribution, damage formation and DNA repair

Recent cancer sequencing efforts have uncovered asymmetry in DNA damage induced mutagenesis between the transcribed and non-transcribed strands of genes. Here, we investigate the major type of damage induced by ultraviolet (UV) radiation, the cyclobutane pyrimidine dimers (CPDs), which are formed primarily in TT dinucleotides. We reveal that a transcriptional asymmetry already exists at the level of TT dinucleotide frequency and therefore also in CPD damage formation. This asymmetry is conserved in vertebrates and invertebrates and is completely reversed between introns and exons. We show the asymmetry in introns is linked to the transcription process itself, and is also found in enhancer elements. In contrast, the asymmetry in exons is not correlated to transcription, and is associated with codon usage preferences. Reanalysis of nucleotide excision repair, normalizing repair to the underlying TT frequencies, we show repair of CPDs is more efficient in exons compared to introns, contributing to the maintenance and integrity of coding regions. Our results highlight the importance of considering the primary sequence of the DNA in determining DNA damage sensitivity and mutagenic potential.

Asymmetron: a toolkit for the identification of strand asymmetry patterns in biological sequences

DNA strand asymmetries can have a major effect on several biological functions, including replication, transcription and transcription factor binding. As such, DNA strand asymmetries and mutational strand bias can provide information about biological function. However, a versatile tool to explore this does not exist. Here, we present Asymmetron, a user-friendly computational tool that performs statistical analysis and visualizations for the evaluation of strand asymmetries. Asymmetron takes as input DNA features provided with strand annotation and outputs strand asymmetries for consecutive occurrences of a single DNA feature or between pairs of features. We illustrate the use of Asymmetron by identifying transcriptional and replicative strand asymmetries of germline structural variant breakpoints. We also show that the orientation of the binding sites of 45% of human transcription factors analyzed have a significant DNA strand bias in transcribed regions, that is also corroborated in ChIP-seq analyses, and is likely associated with transcription. In summary, we provide a novel tool to assess DNA strand asymmetries and show how it can be used to derive new insights across a variety of biological disciplines.

Genome-wide strand asymmetry in massively parallel reporter activity favors genic strands

Massively parallel reporter assays (MPRAs) are useful tools to discover and characterize regulatory elements in human genomes. Partly because enhancer function is assumed to be orientation independent with respect to each strand of the DNA helix, most reported MPRA results ignore stranded information. However, we find pervasive strand asymmetry of MPRA signals in datasets from multiple reporter configurations and in both published and newly reported data. These effects are reproducible across different cell types and in different treatments within a cell type, and are observed both within and outside of annotated regulatory elements. From elements in gene bodies, MPRA strand asymmetry favors the sense strand, suggesting that biological function related to endogenous transcription is driving the phenomenon. Similarly, within Alu mobile element insertions, we find that strand asymmetry favors the transcribed strand of the ancestral retrotransposon. The effect is consistent across the multiplicity of Alu elements in human genomes, and is more pronounced in younger, less diverged Alu elements. We find sequence features driving MPRA strand asymmetry and demonstrate its prediction from sequence alone. We see some evidence for both RNA stabilization and transcriptional activation mechanisms, and hypothesize that the effect is driven by natural selection favoring efficient transcription. Our results indicate that strand asymmetry, as a pervasive and reproducible feature, should be accounted for in analysis of MRPA data. More importantly, the fact that MPRA asymmetry favors naturally transcribed strands suggests that it stems from preserved biological functions that have a substantial, global impact on gene and genome evolution.

Quantifying Influences on Intragenomic Mutation Rate

We report work to quantify the impact on the probability of human genome polymorphism both of recombination and of sequence context at different scales. We use population-based analyses of data on human genetic variants obtained from the public Ensembl database. For recombination, we calculate the variance due to recombination and the probability that a recombination event causes a mutation. We employ novel statistical procedures to take account of the spatial auto-correlation of recombination and mutation rates along the genome. Our results support the view that genomic diversity in recombination hotspots arises largely from a direct effect of recombination on mutation rather than predominantly from the effect of selective sweeps. We also use the statistic of variance due to context to compare the effect on the probability of polymorphism of contexts of various sizes. We find that when the 12 point mutations are considered separately, variance due to context increases significantly as we move from 3-mer to 5-mer and from 5-mer to 7-mer contexts. However, when all mutations are considered in aggregate, these differences are outweighed by the effect of interaction between the central base and its immediate neighbors. This interaction is itself dominated by the transition mutations, including, but not limited to, the CpG effect. We also demonstrate strand-asymmetry of contextual influence in intronic regions, which is hypothesized to be a result of transcription coupled DNA repair. We consider the extent to which the measures we have used can be used to meaningfully compare the relative magnitudes of the impact of recombination and context on mutation.

Architectural instability, inverted skews and mitochondrial phylogenomics of Isopoda: outgroup choice affects the long-branch attraction artefacts

The majority strand of mitochondrial genomes of crustaceans usually exhibits negative GC skews. Most isopods exhibit an inversed strand asymmetry, believed to be a consequence of an inversion of the replication origin (ROI). Recently, we proposed that an additional ROI event in the common ancestor of Cymothoidae and Corallanidae families resulted in a double-inverted skew (negative GC), and that taxa with homoplastic skews cluster together in phylogenetic analyses (long-branch attraction, LBA). Herein, we further explore these hypotheses, for which we sequenced the mitogenome of Asotana magnifica (Cymothoidae), and tested whether our conclusions were biased by poor taxon sampling and inclusion of outgroups. (1) The new mitogenome also exhibits a double-inverted skew, which supports the hypothesis of an additional ROI event in the common ancestor of Cymothoidae and Corallanidae families. (2) It exhibits a unique gene order, which corroborates that isopods possess exceptionally destabilized mitogenomic architecture. (3) Improved taxonomic sampling failed to resolve skew-driven phylogenetic artefacts. (4) The use of a single outgroup exacerbated the LBA, whereas both the use of a large number of outgroups and complete exclusion of outgroups ameliorated it.