Replication Slippage Research Articles

Mitochondrial genomes of blister beetles (Coleoptera, Meloidae) and two large intergenic spacers in Hycleus genera

BackgroundInsect mitochondrial genomes (mitogenomes) exhibit high diversity in some lineages. The gene rearrangement and large intergenic spacer (IGS) have been reported in several Coleopteran species, although very little is known about mitogenomes of Meloidae.ResultsWe determined complete or nearly complete mitogenomes of seven meloid species. The circular genomes encode 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs) and two ribosomal RNAs (rRNAs), and contain a control region, with gene arrangement identical to the ancestral type for insects. The evolutionary rates of all PCGs indicate that their evolution is based on purifying selection. The comparison of tRNA secondary structures indicates diverse substitution patterns in Meloidae. Remarkably, all mitogenomes of the three studied Hycleus species contain two large intergenic spacers (IGSs). IGS1 is located between trnW and trnC, including a 9 bp consensus motif. IGS2 is located between trnS2 (UCN) and nad1, containing discontinuous repeats of a pentanucleotide motif and two 18-bp repeat units in both ends. To date, IGS2 is found only in genera Hycleus across all published Coleopteran mitogenomes. The duplication/random loss model and slipped-strand mispairing are proposed as evolutionary mechanisms for the two IGSs (IGS1, IGS2). The phylogenetic analyses using MrBayes, RAxML, and PhyloBayes methods based on nucleotide and amino acid datasets of 13 PCGs from all published mitogenomes of Tenebrionoids, consistently recover the monophylies of Meloidae and Tenebrionidae. Within Meloidae, the genus Lytta clusters with Epicauta rather than with Mylabris. Although data collected thus far could not resolve the phylogenetic relationships within Meloidae, this study will assist in future mapping of the Meloidae phylogeny.ConclusionsThis study presents mitogenomes of seven meloid beetles. New mitogenomes retain the genomic architecture of the Coleopteran ancestor, but contain two IGSs in the three studied Hycleus species. Comparative analyses of two IGSs suggest that their evolutionary mechanisms are duplication/random loss model and slipped-strand mispairing.

Characterization of the microDNA through the response to chemotherapeutics in lymphoblastoid cell lines.

Recently, a new class of extrachromosomal circular DNA, called microDNA, was identified. They are on average 100 to 400 bp long and are derived from unique non-repetitive genomic regions with high gene density. MicroDNAs are thought to arise from DNA breaks associated with RNA metabolism or replication slippage. Given the paucity of information on this entirely novel phenomenon, we aimed to get an additional insight into microDNA features by performing the microDNA analysis in 20 independent human lymphoblastoid cell lines (LCLs) prior and after treatment with chemotherapeutic drugs. The results showed non-random genesis of microDNA clusters from the active regions of the genome. The size periodicity of 190 bp was observed, which matches DNA fragmentation typical for apoptotic cells. The chemotherapeutic drug-induced apoptosis of LCLs increased both number and size of clusters further suggesting that part of microDNAs could result from the programmed cell death. Interestingly, proportion of identified microDNA sequences has common loci of origin when compared between cell line experiments. While compatible with the original observation that microDNAs originate from a normal physiological process, obtained results imply complementary source of its production. Furthermore, non-random genesis of microDNAs depicted by redundancy between samples makes these entities possible candidates for new biomarker generation.

The Pneumococcal Serotype 15C Capsule Is Partially O-Acetylated and Allows for Limited Evasion of 23-Valent Pneumococcal Polysaccharide Vaccine-Elicited Anti-Serotype 15B Antibodies.

As a species, Streptococcus pneumoniae (the pneumococcus) utilizes a diverse array of capsular polysaccharides to evade the host. In contrast to large variations in sugar composition and linkage formation, O-acetylation is a subtle capsular modification that nonetheless has a large impact on capsular shielding and recognition of the capsule by vaccine-elicited antibodies. Serotype 15B, which is included in the 23-valent pneumococcal polysaccharide vaccine (PPV23), carries the putative O-acetyltransferase gene wciZ The coding sequence of wciZ contains eight consecutive TA repeats [(TA)8]. Replication slippage is thought to result in the addition or loss of TA repeats, subsequently causing frameshift and truncation of WciZ to yield a nonacetylated serotype, 15C. Using sensitive serological tools, we show that serotype 15C isolates whose wciZ contains seven or nine TA repeats retain partial O-acetylation, while serotype 15C isolates whose wciZ contains six TA repeats have barely detectable O-acetylation. We confirmed by inhibition enzyme-linked immunosorbent assay that (TA)7 serotype 15C is ∼0.1% as acetylated as serotype 15B, while serotype 15X is nonacetylated. To eliminate the impact of genetic background, we created isogenic serotype 15B, (TA)7 serotype 15C, and 15BΔwciZ (15X) strains and found that reduction or absence of WciZ-mediated O-acetylation did not affect capsular shielding from phagocytes, biofilm formation, adhesion to nasopharyngeal cells, desiccation tolerance, or murine colonization. Sera from PPV23-immunized persons opsonized serotype 15B significantly but only slightly better than serotypes 15C and 15X; thus, PPV23 may not result in expansion of serotype 15C.

Chimeras Linked to Tandem Repeats and Transposable Elements in Tetraploid Hybrid Fish.

The formation of the allotetraploid hybrid lineage (4nAT) encompasses both distant hybridization and polyploidization processes. The allotetraploid offspring have two sets of sub-genomes inherited from both parental species, and therefore, it is important to explore its genetic structure. Herein, we construct a bacterial artificial chromosome library of allotetraploids, and then sequence and analyze the full-length sequences of 19 bacterial artificial chromosomes. Sixty-eight DNA chimeras are identified, which are divided into four models according to the distribution of the genomic DNA derived from the parents. Among the 68 genetic chimeras, 44 (64.71%) are linked to tandem repeats (TRs) and 23 (33.82%) are linked to transposable elements (TEs). The chimeras linked to TRs are related to slipped-strand mispairing and double-strand break repair while the chimeras linked to TEs benefit from the intervention of recombinases. In addition, TRs and TEs can also result in insertions/deletions of DNA segments. We conclude that DNA chimeras accompanied by TRs and TEs coordinate a balance between the sub-genomes derived from the parents. It is the first report on the relationship between formation of the DNA chimeras and TRs and TEs in the polyploid animals.

In-frame seven amino-acid duplication in AIP arose over the last 3000 years, disrupts protein interaction and stability and is associated with gigantism

ObjectiveMutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene are associated with pituitary adenoma, acromegaly and gigantism. Identical alleles in unrelated pedigrees could be inherited from a common ancestor or result from recurrent mutation events.Design and methodsObservational, inferential and experimental study, including: AIP mutation testing; reconstruction of 14 AIP-region (8.3 Mbp) haplotypes; coalescent-based approximate Bayesian estimation of the time to most recent common ancestor (tMRCA) of the derived allele; forward population simulations to estimate current number of allele carriers; proposal of mutation mechanism; protein structure predictions; co-immunoprecipitation and cycloheximide chase experiments.ResultsNine European-origin, unrelated c.805_825dup-positive pedigrees (four familial, five sporadic from the UK, USA and France) included 16 affected (nine gigantism/four acromegaly/two non-functioning pituitary adenoma patients and one prospectively diagnosed acromegaly patient) and nine unaffected carriers. All pedigrees shared a 2.79 Mbp haploblock around AIP with additional haploblocks privately shared between subsets of the pedigrees, indicating the existence of an evolutionarily recent common ancestor, the ‘English founder’, with an estimated median tMRCA of 47 generations (corresponding to 1175 years) with a confidence interval (9–113 generations, equivalent to 225–2825 years). The mutation occurred in a small tandem repeat region predisposed to slipped strand mispairing. The resulting seven amino-acid duplication disrupts interaction with HSP90 and leads to a marked reduction in protein stability.ConclusionsThe c.805_825dup allele, originating from a common ancestor, associates with a severe clinical phenotype and a high frequency of gigantism. The mutation is likely to be the result of slipped strand mispairing and affects protein–protein interactions and AIP protein stability.

A Dynamic Tandem Repeat in Monocotyledons Inferred from a Comparative Analysis of Chloroplast Genomes in Melanthiaceae.

Chloroplast genomes (cpDNA) are highly valuable resources for evolutionary studies of angiosperms, since they are highly conserved, are small in size, and play critical roles in plants. Slipped-strand mispairing (SSM) was assumed to be a mechanism for generating repeat units in cpDNA. However, research on the employment of different small repeated sequences through SSM events, which may induce the accumulation of distinct types of repeats within the same region in cpDNA, has not been documented. Here, we sequenced two chloroplast genomes from the endemic species Heloniopsis tubiflora (Korea) and Xerophyllum tenax (USA) to cover the gap between molecular data and explore “hot spots” for genomic events in Melanthiaceae. Comparative analysis of 23 complete cpDNA sequences revealed that there were different stages of deletion in the rps16 region across the Melanthiaceae. Based on the partial or complete loss of rps16 gene in cpDNA, we have firstly reported potential molecular markers for recognizing two sections (Veratrum and Fuscoveratrum) of Veratrum. Melathiaceae exhibits a significant change in the junction between large single copy and inverted repeat regions, ranging from trnH_GUG to a part of rps3. Our results show an accumulation of tandem repeats in the rpl23-ycf2 regions of cpDNAs. Small conserved sequences exist and flank tandem repeats in further observation of this region across most of the examined taxa of Liliales. Therefore, we propose three scenarios in which different small repeated sequences were used during SSM events to generate newly distinct types of repeats. Occasionally, prior to the SSM process, point mutation event and double strand break repair occurred and induced the formation of initial repeat units which are indispensable in the SSM process. SSM may have likely occurred more frequently for short repeats than for long repeat sequences in tribe Parideae (Melanthiaceae, Liliales). Collectively, these findings add new evidence of dynamic results from SSM in chloroplast genomes which can be useful for further evolutionary studies in angiosperms. Additionally, genomics events in cpDNA are potential resources for mining molecular markers in Liliales.

Biomechanical Investigation of Different Preparation Techniques of Tripled Soft Tissue ACL Grafts

Aims and Objectives:The purpose was to investigate biomechanical properties of different preparation techniques of tripled soft tissue ACL grafts to avoid early mechanical graft failure. It was hypothesized that 1) a graft with a quadrupled femoral portion results in higher load to failure and stiffness compared to a uniformly tripled graft, and 2) a Krackow stitch technique is superior to both baseball stitch and whipstitch technique.Materials and Methods:Forty-eight bovine flexor tendons (eight per group) with a final graft length of 8 cm were used for the present study and randomly assigned to the two graft configuration groups: A) quadrupled femoral portion with a tripled tibial portion, and B) uniformly tripled soft tissue graft. Within the two groups, three different stitching techniques were applied: 1) Whipstitch technique, 2) Krackow stitch, and 3) Baseball stitch. After preconditioning, the grafts were cyclically loaded for 1000 cycles between 50 and 200 N (1 Hz) with consecutive load to failure testing using a constant elongation of 30 mm/min. During biomechnical testing, graft elongation was evaluated with an optical tracking system. For statistical analysis a two-way analysis of variance with a post-hoc Holm-Sidak test was performed. To evaluate the failure mode the Fisher’s exact test was used. The significance level was set at p #CHR: lesslike# 0.05.Results:Considering load to failure, quadrupling the femoral portion of the graft significantly increased the load to failure (p = 0.003) while varying the stitching technique had no influence (p = 0.998). Additionally, graft elongation was lower in group A, but difference was not significant (p = 0.058). Lowest graft elongation was observed when a Krackow stitch was applied, followed by the baseball stitch, and the whipstitch technique (p = 0.035). In group A, all the constructs (graft including sutures) failed at the suture, whereas in group B the failure mode was a slippage of the free femoral graft strand in seven of eight grafts (p = 0.005).Conclusion:When using tripled ACL soft tissue grafts in combination with an extracortical fixation device, the femoral portion should be quadrupled whereas the stitching technique does not have a significant influence on the biomechanical properties. To achieve a higher load to failure with less graft elongation at time zero, the free femoral strand of the tendon graft should be wrapped around the femoral graft tendon loop. This could reduce early graft failure due to slippage of the third free femoral graft strand.

Evolving Notch polyQ tracts reveal possible solenoid interference elements.

Polyglutamine (polyQ) tracts in regulatory proteins are extremely polymorphic. As functional elements under selection for length, triplet repeats are prone to DNA replication slippage and indel mutations. Many polyQ tracts are also embedded within intrinsically disordered domains, which are less constrained, fast evolving, and difficult to characterize. To identify structural principles underlying polyQ tracts in disordered regulatory domains, here I analyze deep evolution of metazoan Notch polyQ tracts, which can generate alleles causing developmental and neurogenic defects. I show that Notch features polyQ tract turnover that is restricted to a discrete number of conserved “polyQ insertion slots”. Notch polyQ insertion slots are: (i) identifiable by an amphipathic “slot leader” motif; (ii) conserved as an intact C-terminal array in a 1-to-1 relationship with the N-terminal solenoid-forming ankyrin repeats (ARs); and (iii) enriched in carboxamide residues (Q/N), whose sidechains feature dual hydrogen bond donor and acceptor atoms. Correspondingly, the terminal loop and β-strand of each AR feature conserved carboxamide residues, which would be susceptible to folding interference by hydrogen bonding with residues outside the ARs. I thus suggest that Notch polyQ insertion slots constitute an array of AR interference elements (ARIEs). Notch ARIEs would dynamically compete with the delicate serial folding induced by adjacent ARs. Huntingtin, which harbors solenoid-forming HEAT repeats, also possesses a similar number of polyQ insertion slots. These results suggest that intrinsically disordered interference arrays featuring carboxamide and polyQ enrichment may constitute coupled proteodynamic modulators of solenoids.

Microsatellite Instability Pathway and EMAST in Colorectal Cancer.

Microsatellite instability (MSI) refers to the biochemical detection of frameshifted microsatellite sequences from genomic DNA. Genesis of MSI is due to defective DNA mismatch repair (MMR) that fails to correct post DNA replicative slippage mistakes at microsatellites. Most of the estimated 100,000 genomic microsatellites are non-coding; however, ~150-300 microsatellites are coding such that, when frameshifted during the pathogenesis of an MSI tumor, can generate immunogenic neopeptide antigens that limit the growth of tumor and prolong patient survival. In addition to the immune reaction and longer survival, patients with MSI colorectal cancers tend to have poorly differentiated tumors with mucinous features that are located in the right colon. Patients with MSI tumors are more resistant to 5-fluorouracil-based adjuvant chemotherapy but may be responsive to PD-1 immune checkpoint blockade. Specific defects of MMR function not only drive MSI but also elevate microsatellite alterations at selected tetranucleotide repeats that may further modify patient outcome.

Helicobacter pylori BabA in adaptation for gastric colonization.

Helicobacter pylori (H. pylori) as a causative agent of gastric complications, is well adapted for the colonization of gastric mucosa. Although the infectious process depends on several factors, the adhesion to the gastric mucosa is the first and important step. Among several outer membrane proteins, BabA is one of the significant protein involving in many inflammatory processes in addition to its role in the attachment for the persistent colonization. We performed a PubMed search using the key words: “babA”, “pylori”, “gastric complications”, “homologous recombination”, “slipped strand mispairing”; a total of 249 articles were displayed. Of these we mainly focused on articles with the full text in English and published between 2005 and 2016. H. pylori BabA is involved in binding with receptors; however, its synthesis is regulated by phase variation. In this review we confirm that H. pylori babA can be modulated at the molecular and functional levels to adapt to the stress within the gastro-intestinal tract.

The role of break-induced replication in large-scale expansions of (CAG)n/(CTG)n repeats.

Expansions of (CAG)n•(CTG)n trinucleotide repeats are responsible for over a dozen neuromuscular and neurodegenerative disorders. Large-scale expansions are typical for human pedigrees and may be explained by iterative small-scale events such as strand slippage during replication or repair DNA synthesis. Alternatively, a distinct mechanism could lead to a large-scale repeat expansion at a step. To distinguish between these possibilities, we developed a novel experimental system specifically tuned to analyze large-scale expansions of (CAG)n•(CTG)n repeats in Saccharomyces cerevisiae. The median size of repeat expansions was ~60 triplets, though additions in excess of 150 triplets were also observed. Genetic analysis revealed that Rad51, Rad52, Mre11, Pol32, Pif1, and Mus81 and/or Yen1 proteins are required for large-scale expansions, whereas proteins previously implicated in small-scale expansions are not involved. Based on these results, we propose a new model for large-scale expansions based on recovery of replication forks broken at (CAG)n•(CTG)n repeats via break-induced replication.

Unusual structures of CCTG repeats and their participation in repeat expansion.

CCTG repeat expansion in intron 1 of the cellular nucleic acid-binding protein (CNBP) gene has been identified to be the genetic cause of myotonic dystrophy type 2 (DM2). Yet the underlying reasons for the genetic instability in CCTG repeats remain elusive. In recent years, CCTG repeats have been found to form various types of unusual secondary structures including mini-dumbbell (MDB), hairpin and dumbbell, revealing that there is a high structural diversity in CCTG repeats intrinsically. Upon strand slippage, the formation of unusual structures in the nascent strand during DNA replication has been proposed to be the culprit of CCTG repeat expansions. On the one hand, the thermodynamic stability, size, and conformational dynamics of these unusual structures affect the propensity of strand slippage. On the other hand, these structural properties determine whether the unusual structure can successfully escape from DNA repair. In this short overview, we first summarize the recent advances in elucidating the solution structures of CCTG repeats. We then discuss the potential pathways by which these unusual structures bring about variable sizes of repeat expansion, high strand slippage propensity and efficient repair escape.

Expression of the Gene for Autotransporter AutB of Neisseria meningitidis Affects Biofilm Formation and Epithelial Transmigration.

Neisseria meningitidis is a Gram-negative bacterium that resides as a commensal in the upper respiratory tract of humans, but occasionally, it invades the host and causes sepsis and/or meningitis. The bacterium can produce eight autotransporters, seven of which have been studied to some detail. The remaining one, AutB, has not been characterized yet. Here, we show that the autB gene is broadly distributed among pathogenic Neisseria spp. The gene is intact in most meningococcal strains. However, its expression is prone to phase variation due to slipped-strand mispairing at AAGC repeats located within the DNA encoding the signal sequence and is switched off in the vast majority of these strains. Moreover, various genetic disruptions prevent autB expression in most of the strains in which the gene is in phase indicating a strong selection against AutB synthesis. We observed that autB is expressed in two of the strains examined and that AutB is secreted and exposed at the cell surface. Functionality assays revealed that AutB synthesis promotes biofilm formation and delays the passage of epithelial cell layers in vitro. We hypothesize that this autotransporter is produced during the colonization process only in specific niches to facilitate microcolony formation, but its synthesis is switched off probably to evade the immune system and facilitate human tissue invasion.

A Mitochondrial Genome of Rhyparochromidae (Hemiptera: Heteroptera) and a Comparative Analysis of Related Mitochondrial Genomes.

The Rhyparochromidae, the largest family of Lygaeoidea, encompasses more than 1,850 described species, but no mitochondrial genome has been sequenced to date. Here we describe the first mitochondrial genome for Rhyparochromidae: a complete mitochondrial genome of Panaorus albomaculatus (Scott, 1874). This mitochondrial genome is comprised of 16,345 bp, and contains the expected 37 genes and control region. The majority of the control region is made up of a large tandem-repeat region, which has a novel pattern not previously observed in other insects. The tandem-repeats region of P. albomaculatus consists of 53 tandem duplications (including one partial repeat), which is the largest number of tandem repeats among all the known insect mitochondrial genomes. Slipped-strand mispairing during replication is likely to have generated this novel pattern of tandem repeats. Comparative analysis of tRNA gene families in sequenced Pentatomomorpha and Lygaeoidea species shows that the pattern of nucleotide conservation is markedly higher on the J-strand. Phylogenetic reconstruction based on mitochondrial genomes suggests that Rhyparochromidae is not the sister group to all the remaining Lygaeoidea, and supports the monophyly of Lygaeoidea.

Minidumbbell: A New Form of Native DNA Structure

The non-B DNA structures formed by short tandem repeats on the nascent strand during DNA replication have been proposed to be the structural intermediates that lead to repeat expansion mutations. Tetranucleotide TTTA and CCTG repeat expansions have been known to cause reduction in biofilm formation in Staphylococcus aureus and myotonic dystrophy type 2 in human, respectively. In this study, we report the first three-dimensional minidumbbell (MDB) structure formed by natural DNA sequences containing two TTTA or CCTG repeats. The formation of MDB provides possible pathways for strand slippage to occur, which ultimately leads to repair escape and thus expansion mutations. Our result here shows that MDB is a highly compact structure composed of two type II loops. In addition to the typical stabilizing interactions in type II loops, MDB shows extensive stabilizing forces between the two loops, including two distinctive modes of interactions between the minor groove residues. The formation of MDB enriches the structural diversity of natural DNA sequences, reveals the importance of loop-loop interactions in unusual DNA structures, and provides insights into novel mechanistic pathways of DNA repeat expansion mutations.

Microsatellite (SSR) amplification by PCR usually led to polymorphic bands: Evidence which shows replication slippage occurs in extend or nascent DNA strands.

Microsatellites or simple sequence repeats (SSRs) are very effective molecular markers in population genetics, genome mapping, taxonomic study and other large-scale studies. Variation in number of tandem repeats within microsatellite refers to simple sequence length polymorphism (SSLP); but there are a few studies that are showed SSRs replication slippage may be occurred during in vitro amplification which are produced 'stutter products' differing in length from the main products. The purpose of this study is introducing a reliable method to realize SSRs replication slippage. At first, three unique primers designed to amplify SSRs loci in the great gerbil (Rhombomys opimus) by PCR. Crush and soak method used to isolate interesting DNA bands from polyacrylamide gel. PCR products analyzed using by sequencing methods. Our study has been shown that Taq DNA polymerase slipped during microsatellite in vitro amplification which led to insertion or deletion of repeats in sense or antisense DNA strands. It is produced amplified fragments with various lengths in gel electrophoresis showed as 'stutter bands'. Thus, in population studies by SSRs markers recommend that replication slippage effects and stutter bands have been considered.

Comparative Methylome Analysis of the Occasional Ruminant Respiratory Pathogen Bibersteinia trehalosi.

We examined and compared both the methylomes and the modification-related gene content of four sequenced strains of Bibersteinia trehalosi isolated from the nasopharyngeal tracts of Nebraska cattle with symptoms of bovine respiratory disease complex. The methylation patterns and the encoded DNA methyltransferase (MTase) gene sets were different between each strain, with the only common pattern being that of Dam (GATC). Among the observed patterns were three novel motifs attributable to Type I restriction-modification systems. In some cases the differences in methylation patterns corresponded to the gain or loss of MTase genes, or to recombination at target recognition domains that resulted in changes of enzyme specificity. However, in other cases the differences could be attributed to differential expression of the same MTase gene across strains. The most obvious regulatory mechanism responsible for these differences was slipped strand mispairing within short sequence repeat regions. The combined action of these evolutionary forces allows for alteration of different parts of the methylome at different time scales. We hypothesize that pleiotropic transcriptional modulation resulting from the observed methylomic changes may be involved with the switch between the commensal and pathogenic states of this common member of ruminant microflora.

TALE-Like Effectors Are an Ancestral Feature of the Ralstonia solanacearum Species Complex and Converge in DNA Targeting Specificity.

Ralstonia solanacearum, a species complex of bacterial plant pathogens divided into four monophyletic phylotypes, causes plant diseases in tropical climates around the world. Some strains exhibit a broad host range on solanaceous hosts, while others are highly host-specific as for example some banana-pathogenic strains. Previous studies showed that transcription activator-like (TAL) effectors from Ralstonia, termed RipTALs, are capable of activating reporter genes in planta, if these are preceded by a matching effector binding element (EBE). RipTALs target DNA via their central repeat domain (CRD), where one repeat pairs with one DNA-base of the given EBE. The repeat variable diresidue dictates base repeat specificity in a predictable fashion, known as the TALE code. In this work, we analyze RipTALs across all phylotypes of the Ralstonia solanacearum species complex. We find that RipTALs are prevalent in phylotypes I and IV but absent from most phylotype III and II strains (10/12, 8/14, 1/24, and 1/5 strains contained a RipTAL, respectively). RipTALs originating from strains of the same phylotype show high levels of sequence similarity (>98%) in the N-terminal and C-terminal regions, while RipTALs isolated from different phylotypes show 47–91% sequence similarity in those regions, giving rise to four RipTAL classes. We show that, despite sequence divergence, the base preference for guanine, mediated by the N-terminal region, is conserved across RipTALs of all classes. Using the number and order of repeats found in the CRD, we functionally sub-classify RipTALs, introduce a new simple nomenclature, and predict matching EBEs for all seven distinct RipTALs identified. We experimentally study RipTAL EBEs and uncover that some RipTALs are able to target the EBEs of other RipTALs, referred to as cross-reactivity. In particular, RipTALs from strains with a broad host range on solanaceous hosts cross-react on each other’s EBEs. Investigation of sequence divergence between RipTAL repeats allows for a reconstruction of repeat array biogenesis, for example through slipped strand mispairing or gene conversion. Using these studies we show how RipTALs of broad host range strains evolved convergently toward a shared target sequence. Finally, we discuss the differences between TALE-likes of plant pathogens in the context of disease ecology.

Repair of adjacent single-strand breaks is often accompanied by the formation of tandem sequence duplications in plant genomes

Duplication of existing sequences is a major mechanism of genome evolution. It has been previously shown that duplications can occur by replication slippage, unequal sister chromatid exchange, homologous recombination, and aberrant double-strand break-induced synthesis-dependent strand annealing reactions. In a recent study, the abundant presence of short direct repeats was documented by comparative bioinformatics analysis of different rice genomes, and the hypothesis was put forward that such duplications might arise due to the concerted repair of adjacent single-strand breaks (SSBs). Applying the CRISPR/Cas9 technology, we were able to test this hypothesis experimentally in the model plant Arabidopsis thaliana Using a Cas9 nickase to induce adjacent genomic SSBs in different regions of the genome (genic, intergenic, and heterochromatic) and at different distances (∼20, 50, and 100 bps), we analyzed the repair outcomes by deep sequencing. In addition to deletions, we regularly detected the formation of direct repeats close to the break sites, independent of the genomic context. The formation of these duplications as well as deletions may be associated with the presence of microhomologies. Most interestingly, we found that even the induction of two SSBs on the same DNA strand can cause genome alterations, albeit at a much lower level. Because such a scenario reflects a natural step during nucleotide excision repair, and given that the germline is set aside only late during development in plants, the repair of adjacent SSBs indeed seems to have an important influence on the shaping of plant genomes during evolution.

Selection pressure on human STR loci and its relevance in repeat expansion disease.

Short Tandem Repeats (STRs) comprise repeats of one to several base pairs. Because of the high mutability due to strand slippage during DNA synthesis, rapid evolutionary change in the number of repeating units directly shapes the range of repeat-number variation according to selection pressure. However, the remaining questions include: Why are STRs causing repeat expansion diseases maintained in the human population; and why are these limited to neurodegenerative diseases? By evaluating the genome-wide selection pressure on STRs using the database we constructed, we identified two different patterns of relationship in repeat-number polymorphisms between DNA and amino-acid sequences, although both patterns are evolutionary consequences of avoiding the formation of harmful long STRs. First, a mixture of degenerate codons is represented in poly-proline (poly-P) repeats. Second, long poly-glutamine (poly-Q) repeats are favored at the protein level; however, at the DNA level, STRs encoding long poly-Qs are frequently divided by synonymous SNPs. Furthermore, significant enrichments of apoptosis and neurodevelopment were biological processes found specifically in genes encoding poly-Qs with repeat polymorphism. This suggests the existence of a specific molecular function for polymorphic and/or long poly-Q stretches. Given that the poly-Qs causing expansion diseases were longer than other poly-Qs, even in healthy subjects, our results indicate that the evolutionary benefits of long and/or polymorphic poly-Q stretches outweigh the risks of long CAG repeats predisposing to pathological hyper-expansions. Molecular pathways in neurodevelopment requiring long and polymorphic poly-Q stretches may provide a clue to understanding why poly-Q expansion diseases are limited to neurodegenerative diseases.