DNA Sequence Context Research Articles

Mitochondrial DNA sequence context in the penetrance of mitochondrial t-RNA mutations: A study across multiple lineages with diagnostic implications.

Mitochondrial DNA (mtDNA) mutations are well recognized as an important cause of inherited disease. Diseases caused by mtDNA mutations exhibit a high degree of clinical heterogeneity with a complex genotype-phenotype relationship, with many such mutations exhibiting incomplete penetrance. There is evidence that the spectrum of mutations causing mitochondrial disease might differ between different mitochondrial lineages (haplogroups) seen in different global populations. This would point to the importance of sequence context in the expression of mutations. To explore this possibility, we looked for mutations which are known to cause disease in humans, in animals of other species unaffected by mtDNA disease. The mt-tRNA genes are the location of many pathogenic mutations, with the m.3243A>G mutation on the mt-tRNA-Leu(UUR) being the most frequently seen mutation in humans. This study looked for the presence of m.3243A>G in 2784 sequences from 33 species, as well as any of the other mutations reported in association with disease located on mt-tRNA-Leu(UUR). We report a number of disease associated variations found on mt-tRNA-Leu(UUR) in other chordates, as the major population variant, with m.3243A>G being seen in 6 species. In these, we also found a number of mutations which appear compensatory and which could prevent the pathogenicity associated with this change in humans. This work has important implications for the discovery and diagnosis of mtDNA mutations in non-European populations. In addition, it might provide a partial explanation for the conflicting results in the literature that examines the role of mtDNA variants in complex traits.

Exploring background mutational processes to decipher cancer genetic heterogeneity.

Much remains unknown about the progression and heterogeneity of mutational processes in different cancers and their diagnostic and clinical potential. A growing body of evidence supports mutation rate dependence on the local DNA sequence context for various types of mutations. We propose several tools for the analysis of cancer context-dependent mutations, which are implemented in an online computational framework MutaGene. The framework explores DNA context-dependent mutational patterns and underlying somatic cancer mutagenesis, analyzes mutational profiles of cancer samples, identifies the combinations of underlying mutagenic processes including those related to infidelity of DNA replication and repair machinery, and various other endogenous and exogenous mutagenic factors. As a result, the combination of mutagenic processes can be identified in any query sample with subsequent comparison to mutational profiles derived from malignant and benign samples. In addition, mutagen or cancer-specific mutational background models are applied to calculate expected DNA and protein site mutability to decouple relative contributions of mutagenesis and selection in carcinogenesis, thus elucidating the site-specific driving events in cancer. MutaGene is freely available at https://www.ncbi.nlm.nih.gov/projects/mutagene/.

Integration site selection by retroviruses and transposable elements in eukaryotes.

Transposable elements and retroviruses are found in most genomes, can be pathogenic and are widely used as gene-delivery and functional genomics tools. Exploring whether these genetic elements target specific genomic sites for integration and how this preference is achieved is crucial to our understanding of genome evolution, somatic genome plasticity in cancer and ageing, host-parasite interactions and genome engineering applications. High-throughput profiling of integration sites by next-generation sequencing, combined with large-scale genomic data mining and cellular or biochemical approaches, has revealed that the insertions are usually non-random. The DNA sequence, chromatin and nuclear context, and cellular proteins cooperate in guiding integration in eukaryotic genomes, leading to a remarkable diversity of insertion site distribution and evolutionary strategies.

Mobile small RNAs regulate genome-wide DNA methylation.

RNA silencing at the transcriptional and posttranscriptional levels regulates endogenous gene expression, controls invading transposable elements (TEs), and protects the cell against viruses. Key components of the mechanism are small RNAs (sRNAs) of 21-24 nt that guide the silencing machinery to their nucleic acid targets in a nucleotide sequence-specific manner. Transcriptional gene silencing is associated with 24-nt sRNAs and RNA-directed DNA methylation (RdDM) at cytosine residues in three DNA sequence contexts (CG, CHG, and CHH). We previously demonstrated that 24-nt sRNAs are mobile from shoot to root in Arabidopsis thaliana and confirmed that they mediate DNA methylation at three sites in recipient cells. In this study, we extend this finding by demonstrating that RdDM of thousands of loci in root tissues is dependent upon mobile sRNAs from the shoot and that mobile sRNA-dependent DNA methylation occurs predominantly in non-CG contexts. Mobile sRNA-dependent non-CG methylation is largely dependent on the DOMAINS REARRANGED METHYLTRANSFERASES 1/2 (DRM1/DRM2) RdDM pathway but is independent of the CHROMOMETHYLASE (CMT)2/3 DNA methyltransferases. Specific superfamilies of TEs, including those typically found in gene-rich euchromatic regions, lose DNA methylation in a mutant lacking 22- to 24-nt sRNAs (dicer-like 2, 3, 4 triple mutant). Transcriptome analyses identified a small number of genes whose expression in roots is associated with mobile sRNAs and connected to DNA methylation directly or indirectly. Finally, we demonstrate that sRNAs from shoots of one accession move across a graft union and target DNA methylation de novo at normally unmethylated sites in the genomes of root cells from a different accession.

Bacterial DNA Methylation and Methylomes.

Formation of C5-methylcytosine, N4-methylcytosine, and N6-methyladenine in bacterial genomes is postreplicative and involves transfer of a methyl group from S-adenosyl-methionine to a base embedded in a specific DNA sequence context. Most bacterial DNA methyltransferases belong to restriction-modification systems; in addition, "solitary" or "orphan" DNA methyltransferases are frequently found in the genomes of bacteria and phage. Base methylation can affect the interaction of DNA-binding proteins with their cognate sites, either by a direct effect (e.g., steric hindrance) or by changes in DNA topology. In both Alphaproteobacteria and Gammaproteobacteria, the roles of DNA base methylation are especially well known for N6-methyladenine, including control of chromosome replication, nucleoid segregation, postreplicative correction of DNA mismatches, cell cycle-coupled transcription, formation of bacterial cell lineages, and regulation of bacterial virulence. Technical procedures that permit genome-wide analysis of DNA methylation are nowadays expanding our knowledge of the extent, evolution, and physiological significance of bacterial DNA methylation.

CG gene body DNA methylation changes and evolution of duplicated genes in cassava

DNA methylation is important for the regulation of gene expression and the silencing of transposons in plants. Here we present genome-wide methylation patterns at single-base pair resolution for cassava (Manihot esculenta, cultivar TME 7), a crop with a substantial impact in the agriculture of subtropical and tropical regions. On average, DNA methylation levels were higher in all three DNA sequence contexts (CG, CHG, and CHH, where H equals A, T, or C) than those of the most well-studied model plant Arabidopsis thaliana. As in other plants, DNA methylation was found both on transposons and in the transcribed regions (bodies) of many genes. Consistent with these patterns, at least one cassava gene copy of all of the known components of Arabidopsis DNA methylation pathways was identified. Methylation of LTR transposons (GYPSY and COPIA) was found to be unusually high compared with other types of transposons, suggesting that the control of the activity of these two types of transposons may be especially important. Analysis of duplicated gene pairs resulting from whole-genome duplication showed that gene body DNA methylation and gene expression levels have coevolved over short evolutionary time scales, reinforcing the positive relationship between gene body methylation and high levels of gene expression. Duplicated genes with the most divergent gene body methylation and expression patterns were found to have distinct biological functions and may have been under natural or human selection for cassava traits.

Mechanism of aromatic amine carcinogen bypass by the Y-family polymerase, Dpo4.

Bulky DNA damage inhibits DNA synthesis by replicative polymerases and often requires the action of error prone bypass polymerases. The exact mechanism governing adduct-induced mutagenesis and its dependence on the DNA sequence context remains unclear. In this work, we characterize Dpo4 binding conformations and activity with DNA templates modified with the carcinogenic DNA adducts, 2-aminofluoene (AF) or N-acetyl-2-aminofluorene (AAF), using single-molecule FRET (smFRET) analysis and DNA synthesis extension assays. We find that in the absence of dNTPs, both adducts alter polymerase binding as measured by smFRET, but the addition of dNTPs induces the formation of a ternary complex having what appears to be a conformation similar to the one observed with an unmodified DNA template. We also observe that the misincorporation pathways for each adduct present significant differences: while an AF adduct induces a structure consistent with the previously observed primer-template looped structure, its acetylated counterpart uses a different mechanism, one consistent with a dNTP-stabilized misalignment mechanism.

Genome-Wide Epigenetic Regulation of Gene Transcription in Maize Seeds.

BackgroundEpigenetic regulation is well recognized for its importance in gene expression in organisms. DNA methylation, an important epigenetic mark, has received enormous attention in recent years as it’s a key player in many biological processes. It remains unclear how DNA methylation contributes to gene transcription regulation in maize seeds. Here, we take advantage of recent technologies to examine the genome-wide association of DNA methylation with transcription of four types of DNA sequences, including protein-coding genes, pseudogenes, transposable elements, and repeats in maize embryo and endosperm, respectively.ResultsThe methylation in CG, CHG and CHH contexts plays different roles in the control of gene expression. Methylation around the transcription start sites and transcription stop regions of protein-coding genes is negatively correlated, but in gene bodies positively correlated, to gene expression level. The upstream regions of protein-coding genes are enriched with 24-nt siRNAs and contain high levels of CHH methylation, which is correlated to gene expression level. The analysis of sequence content within CG, CHG, or CHH contexts reveals that only CHH methylation is affected by its local sequences, which is different from Arabidopsis.ConclusionsIn summary, we conclude that methylation-regulated transcription varies with the types of DNA sequences, sequence contexts or parts of a specific gene in maize seeds and differs from that in other plant species. Our study helps people better understand from a genome-wide viewpoint that how transcriptional expression is controlled by DNA methylation, one of the important factors influencing transcription, and how the methylation is associated with small RNAs.

Base damage, local sequence context and TP53 mutation hotspots: a molecular dynamics study of benzo[a]pyrene induced DNA distortion and mutability.

The mutational pattern for the TP53 tumour suppressor gene in lung tumours differs to other cancer types by having a higher frequency of G:C>T:A transversions. The aetiology of this differing mutation pattern is still unknown. Benzo[a]pyrene,diol epoxide (BPDE) is a potent cigarette smoke carcinogen that forms guanine adducts at TP53 CpG mutation hotspot sites including codons 157, 158, 245, 248 and 273. We performed molecular modelling of BPDE-adducted TP53 duplex sequences to determine the degree of local distortion caused by adducts which could influence the ability of nucleotide excision repair. We show that BPDE adducted codon 157 has greater structural distortion than other TP53 G:C>T:A hotspot sites and that sequence context more distal to adjacent bases must influence local distortion. Using TP53 trinucleotide mutation signatures for lung cancer in smokers and non-smokers we further show that codons 157 and 273 have the highest mutation probability in smokers. Combining this information with adduct structural data we predict that G:C>T:A mutations at codon 157 in lung tumours of smokers are predominantly caused by BPDE. Our results provide insight into how different DNA sequence contexts show variability in DNA distortion at mutagen adduct sites that could compromise DNA repair at well characterized cancer related mutation hotspots.

High-resolution Digital Mapping of N-Methylpurines in Human Cells Reveals Modulation of Their Induction and Repair by Nearest-neighbor Nucleotides

N-Methylpurines (NMPs), including N(7)-methylguanine (7MeG) and N(3)-methyladenine (3MeA), can be induced by environmental methylating agents, chemotherapeutics, and natural cellular methyl donors. In human cells, NMPs are repaired by the multi-step base excision repair pathway initiated by human alkyladenine glycosylase. Repair of NMPs has been shown to be affected by DNA sequence contexts. However, the nature of the sequence contexts has been poorly understood. We developed a sensitive method, LAF-Seq (Lesion-Adjoining Fragment Sequencing), which allows nucleotide-resolution digital mapping of DNA damage and repair in multiple genomic fragments of interest in human cells. We also developed a strategy that allows accurate measurement of the excision kinetics of NMP bases in vitro. We demonstrate that 3MeAs are induced to a much lower level by the SN2 methylating agent dimethyl sulfate and repaired much faster than 7MeGs in human fibroblasts. Induction of 7MeGs by dimethyl sulfate is affected by nearest-neighbor nucleotides, being enhanced at sites neighbored by a G or T on the 3' side, but impaired at sites neighbored by a G on the 5' side. Repair of 7MeGs is also affected by nearest-neighbor nucleotides, being slow if the lesions are between purines, especially Gs, and fast if the lesions are between pyrimidines, especially Ts. Excision of 7MeG bases from the DNA backbone by human alkyladenine glycosylase in vitro is similarly affected by nearest-neighbor nucleotides, suggesting that the effect of nearest-neighbor nucleotides on repair of 7MeGs in the cells is primarily achieved by modulating the initial step of the base excision repair process.

DNA sequence context greatly affects the accuracy of bypass across an ultraviolet light 6-4 photoproduct in mammalian cells

Translesion DNA synthesis (TLS) is a DNA damage tolerance mechanism carried out by low-fidelity DNA polymerases that bypass DNA lesions, which overcomes replication stalling. Despite the miscoding nature of most common DNA lesions, several of them are bypassed in mammalian cells in a relatively accurate manner, which plays a key role maintaining a low mutation load. Whereas it is generally agreed that TLS across the major UV and sunlight induced DNA lesion, the cyclobutane pyrimidine dimer (CPD), is accurate, there were conflicting reports on whether the same is true for the thymine–thymine pyrimidine–pyrimidone(6-4) ultraviolet light photoproduct (TT6-4PP), which represents the second most common class of UV lesions. Using a TLS assay system based on gapped plasmids carrying site-specific TT6-4PP lesions in defined sequence contexts we show that the DNA sequence context markedly affected both the extent and accuracy of TLS. The sequence exhibiting higher TLS exhibited also higher error-frequency, caused primarily by semi-targeted mutations, at the nearest nucleotides flanking the lesion. Our results resolve the discrepancy reported on TLS across TT6-4PP, and suggest that TLS is more accurate in human cells than in mouse cells.

Repair efficiency of clustered abasic sites by APE1 in nucleosome core particles is sequence and position dependent

DNA sequence context and nucleosomal positioning guide the repair efficiency of clustered abasic sites by APE1 enzyme.

H3K4me1 marks DNA regions hypomethylated during aging in human stem and differentiated cells.

In differentiated cells, aging is associated with hypermethylation of DNA regions enriched in repressive histone post-translational modifications. However, the chromatin marks associated with changes in DNA methylation in adult stem cells during lifetime are still largely unknown. Here, DNA methylation profiling of mesenchymal stem cells (MSCs) obtained from individuals aged 2 to 92 yr identified 18,735 hypermethylated and 45,407 hypomethylated CpG sites associated with aging. As in differentiated cells, hypermethylated sequences were enriched in chromatin repressive marks. Most importantly, hypomethylated CpG sites were strongly enriched in the active chromatin mark H3K4me1 in stem and differentiated cells, suggesting this is a cell type–independent chromatin signature of DNA hypomethylation during aging. Analysis of scedasticity showed that interindividual variability of DNA methylation increased during aging in MSCs and differentiated cells, providing a new avenue for the identification of DNA methylation changes over time. DNA methylation profiling of genetically identical individuals showed that both the tendency of DNA methylation changes and scedasticity depended on nongenetic as well as genetic factors. Our results indicate that the dynamics of DNA methylation during aging depend on a complex mixture of factors that include the DNA sequence, cell type, and chromatin context involved and that, depending on the locus, the changes can be modulated by genetic and/or external factors.

Replicative bypass of abasic site in Escherichia coli and human cells: similarities and differences.

Abasic [apurinic/apyrimidinic (AP)] sites are the most common DNA damages, opposite which dAMP is frequently inserted (‘A-rule’) in Escherichia coli. Nucleotide insertion opposite the AP-site in eukaryotic cells depends on the assay system and the type of cells. Accordingly, a ‘C-rule’, ‘A-rule’, or the lack of specificity has been reported. DNA sequence context also modulates nucleotide insertion opposite AP-site. Herein, we have compared replication of tetrahydrofuran (Z), a stable analog of AP-site, in E. coli and human embryonic kidney 293T cells in two different sequences. The efficiency of translesion synthesis or viability of the AP-site construct in E. coli was less than 1%, but it was 7- to 8-fold higher in the GZGTC sequence than in the GTGZC sequence. The difference in viability increased even more in pol V-deficient strains. Targeted one-base deletions occurred in 63% frequency in the GZG and 68% frequency in GZC sequence, which dropped to 49% and 21%, respectively, upon induction of SOS. The full-length products with SOS primarily involved dAMP insertion opposite the AP-site, which occurred in 49% and 71% frequency, respectively, in the GZG and GZC sequence. dAMP insertion, largely carried out by pol V, was more efficient when the AP-site was a stronger replication block. In contrast to these results in E. coli, viability was 2 to 3 orders of magnitude higher in human cells, and the ‘A-rule’ was more rigidly followed. The AP-site in the GZG and GZC sequences gave 76% and 89%, respectively, Z→T substitutions. In human cells, targeted one-base deletion was undetectable, and dTMP>dCMP were the next preferred nucleotides inserted opposite Z. siRNA knockdown of Rev1 or pol ζ established that both these polymerases are vital for AP-site bypass, as demonstrated by 36–67% reduction in bypass efficiency. However, neither polymerase was indispensable, suggesting roles of additional DNA polymerases in AP-site bypass in human cells.

Modeling DNA methylation dynamics with approaches from phylogenetics.

Motivation: Methylation of CpG dinucleotides is a prevalent epigenetic modification that is required for proper development in vertebrates. Genome-wide DNA methylation assays have become increasingly common, and this has enabled characterization of DNA methylation in distinct stages across differentiating cellular lineages. Changes in CpG methylation are essential to cellular differentiation; however, current methods for modeling methylation dynamics do not account for the dependency structure between precursor and dependent cell types.Results: We developed a continuous-time Markov chain approach, based on the observation that changes in methylation state over tissue differentiation can be modeled similarly to DNA nucleotide changes over evolutionary time. This model explicitly takes precursor to descendant relationships into account and enables inference of CpG methylation dynamics. To illustrate our method, we analyzed a high-resolution methylation map of the differentiation of mouse stem cells into several blood cell types. Our model can successfully infer unobserved CpG methylation states from observations at the same sites in related cell types (90% correct), and this approach more accurately reconstructs missing data than imputation based on neighboring CpGs (84% correct). Additionally, the single CpG resolution of our methylation dynamics estimates enabled us to show that DNA sequence context of CpG sites is informative about methylation dynamics across tissue differentiation. Finally, we identified genomic regions with clusters of highly dynamic CpGs and present a likely functional example. Our work establishes a framework for inference and modeling that is well suited to DNA methylation data, and our success suggests that other methods for analyzing DNA nucleotide substitutions will also translate to the modeling of epigenetic phenomena.Availability and implementation: Source code is available at www.kostkalab.net/software.Contact: tony.capra@vanderbilt.edu or kostka@pitt.edu

O⁶-carboxymethylguanine in DNA forms a sequence context-dependent wobble base-pair structure with thymine.

N-Nitrosation of glycine and its derivatives generates potent alkylating agents that can lead to the formation of O(6)-carboxymethylguanine (O(6)-CMG) in DNA. O(6)-CMG has been identified in DNA derived from human colon tissue and its occurrence has been linked to diets high in red and processed meats, implying an association with the induction of colorectal cancer. By analogy to O(6)-methylguanine, O(6)-CMG is expected to be mutagenic, inducing G-to-A mutations that may be the molecular basis of increased cancer risk. Previously, the crystal structure of the DNA dodecamer d(CGCG[O(6)-CMG]ATTCGCG) has been reported, in which O(6)-CMG forms a Watson-Crick-type pair with thymine similar to the canonical A:T pair. In order to further investigate the versatility of O(6)-CMG in base-pair formation, the structure of the DNA dodecamer d(CGC[O(6)-CMG]AATTTGCG) containing O(6)-CMG at a different position has been determined by X-ray crystallography using four crystal forms obtained under conditions containing different solvent ions (Sr(2+), Ba(2+), Mg(2+), K(+) or Na(+)) with and without Hoechst 33258. The most striking finding is that the pairing modes of O(6)-CMG with T are quite different from those previously reported. In the present dodecamer, the T bases are displaced (wobbled) into the major groove to form a hydrogen bond between the thymine N(3) N-H and the carboxyl group of O(6)-CMG. In addition, a water molecule is bridged through two hydrogen bonds between the thymine O(2) atom and the 2-amino group of O(6)-CMG to stabilize the pairing. These interaction modes commonly occur in the four crystal forms, regardless of the differences in crystallization conditions. The previous and the present results show that O(6)-CMG can form a base pair with T in two alternative modes: the Watson-Crick type and a high-wobble type, the nature of which may depend on the DNA-sequence context.

Immunoglobulin somatic hypermutation by APOBEC3/Rfv3 during retroviral infection.

Somatic hypermutation (SHM) is an integral process in the development of high-affinity antibodies that are important for recovery from viral infections and vaccine-induced protection. Ig SHM occurs predominantly in germinal centers (GC) via the enzymatic activity of activation-induced deaminase (AID). In contrast, the evolutionarily related apolipoprotein B mRNA-editing enzyme, catalytic polypeptide 3 (APOBEC3) proteins are known to restrict retroviruses, including HIV-1. We previously reported that mouse APOBEC3 encodes Recovery from Friend virus 3 (Rfv3), a classical resistance gene in mice that promotes the neutralizing antibody response against retrovirus infection. We now show that APOBEC3/Rfv3 complements AID in driving Ig SHM during retrovirus infection. Analysis of antibody sequences from retrovirus-specific hybridomas and GC B cells from infected mice revealed Ig heavy-chain V genes with significantly increased C-to-T and G-to-A transitions in wild-type as compared with APOBEC3-defective mice. The context of the mutations was consistent with APOBEC3 but not AID mutational activity. These findings help explain the role of APOBEC3/Rfv3 in promoting the neutralizing antibody responses essential for recovery from retroviral infection and highlight APOBEC3-mediated deamination as a previously unidentified mechanism for antibody diversification in vivo.

One-pot approach for examining the DNA methylation patterns using an engineered methyl-probe

Aberrant DNA methylation is a common observation in various types of human cancers, i.e., breast and lung cancers. Nevertheless, the current DNA methylation detection approaches require bisulfite treatments and are laborious or costly to perform. To address these challenges, we developed a methyl-probe based on the MBD1 protein. Combined with fluorescence correlation spectroscopy, our probe can sensitively detect the existence of DNA methylation at concentrations above 20nM in a one-pot assay. The probe can quantify the total amount of methylated CG dinucleotides above ~20nM, independent of DNA sequence contexts, concentrations (20–1900nM) and methylation levels (5–100%). Our detection platform offers a simple and cheap alternative DNA methylation detection approach.

Structure and mutagenesis of the DNA modification-dependent restriction endonuclease AspBHI

The modification-dependent restriction endonuclease AspBHI recognizes 5-methylcytosine (5mC) in the double-strand DNA sequence context of (C/T)(C/G)(5mC)N(C/G) (N = any nucleotide) and cleaves the two strands a fixed distance (N12/N16) 3′ to the modified cytosine. We determined the crystal structure of the homo-tetrameric AspBHI. Each subunit of the protein comprises two domains: an N-terminal DNA-recognition domain and a C-terminal DNA cleavage domain. The N-terminal domain is structurally similar to the eukaryotic SET and RING-associated (SRA) domain, which is known to bind to a hemi-methylated CpG dinucleotide. The C-terminal domain is structurally similar to classic Type II restriction enzymes and contains the endonuclease catalytic-site motif of DX20EAK. To understand how specific amino acids affect AspBHI recognition preference, we generated a homology model of the AspBHI-DNA complex, and probed the importance of individual amino acids by mutagenesis. Ser41 and Arg42 are predicted to be located in the DNA minor groove 5′ to the modified cytosine. Substitution of Ser41 with alanine (S41A) and cysteine (S41C) resulted in mutants with altered cleavage activity. All 19 Arg42 variants resulted in loss of endonuclease activity.

Epigenetic landscape for initiation of DNA replication

The key genetic process of DNA replication is initiated at specific sites referred to as replication origins. In eukaryotes, origins of DNA replication are not specified by a defined nucleotide sequence. Recent studies have shown that the structural context and topology of DNA sequence, chromatin features, and its transcriptional activity play an important role in origin choice. During differentiation and development, significant changes in chromatin organization and transcription occur, influencing origin activity and choice. In the last few years, a number of different genome-wide studies have broadened the understanding of replication origin regulation. In this review, we discuss the epigenetic factors and mechanisms that modulate origin choice and firing.