Xenopus Borealis Research Articles

Preparation of Xenopus borealis and Xenopus tropicalis Egg Extracts for Comparative Cell Biology and Evolutionary Studies.

Cytoplasmic extracts prepared from eggs of the African clawed frog Xenopus laevis are extensively used to study various cellular events including the cell cycle, cytoskeleton dynamics, and cytoplasm organization, as well as the biology of membranous organelles and phase-separated non-membrane-bound structures. Recent development of extracts from eggs of other Xenopus allows interspecies comparisons that provide new insights into morphological and biological size variations and underlying mechanisms across evolution. Here, we describe methods to prepare cytoplasmic extracts from eggs of the allotetraploid Marsabit clawed frog, Xenopus borealis, and the diploid Western clawed frog, Xenopus tropicalis. We detail mixing and "hybrid" experiments that take advantage of the physiological but highly accessible nature of extracts to reveal the evolutionary relationships across species. These new developments create a robust and versatile toolbox to elucidate molecular, cell biological, and evolutionary questions in essential cellular processes.

New insights into Xenopus sex chromosome genomics from the Marsabit clawed frog X.borealis.

In many groups, sex chromosomes change frequently but the drivers of their rapid evolution are varied and often poorly characterized. With an aim of further understanding sex chromosome turnover, we investigated the polymorphic sex chromosomes of the Marsabit clawed frog, Xenopus borealis, using genomic data and a new chromosome-scale genome assembly. We confirmed previous findings that 54.1Mb of chromosome 8L is sex-linked in animals from east Kenya and a laboratory strain, but most (or all) of this region is not sex-linked in natural populations from west Kenya. Previous work suggests possible degeneration of the Z chromosomes in the east population because many sex-linked transcripts of this female heterogametic population have female-biased expression, and we therefore expected this chromosome to not be present in the west population. In contrast, our simulations support a model where most or all of the sex-linked portion of the Z chromosome from the east acquired autosomal segregation in the west, and where much genetic variation specific to the large sex-linked portion of the W chromosome from the east is not present in the west. These recent changes are consistent with the hot-potato model, wherein sex chromosome turnover is favoured by natural selection if it purges a (minimally) degenerate sex-specific sex chromosome, but counterintuitively suggest natural selection failed to purge a Z chromosome that has signs of more advanced and possibly more ancient regulatory degeneration. These findings highlight complex evolutionary dynamics of young, rapidly evolving Xenopus sex chromosomes and set the stage for mechanistic work aimed at pinpointing additional sex-determining genes in this group.

Spindle assembly in egg extracts of the Marsabit clawed frog, Xenopus borealis.

Egg extracts of the African clawed frog Xenopus laevis have provided a cell-free system instrumental in elucidating events of the cell cycle, including mechanisms of spindle assembly. Comparison with extracts from the diploid Western clawed frog, Xenopus tropicalis, which is smaller at the organism, cellular and subcellular levels, has enabled the identification of spindle size scaling factors. We set out to characterize the Marsabit clawed frog, Xenopus borealis, which is intermediate in size between the two species, but more recently diverged in evolution from X. laevis than X. tropicalis. X. borealis eggs were slightly smaller than those of X. laevis, and slightly smaller spindles were assembled in egg extracts. Interestingly, microtubule distribution across the length of the X. borealis spindles differed from both X. laevis and X. tropicalis. Extract mixing experiments revealed common scaling phenomena among Xenopus species, while characterization of spindle factors katanin, TPX2, and Ran indicate that X. borealis spindles possess both X. laevis and X. tropicalis features. Thus, X. borealis egg extract provides a third in vitro system to investigate interspecies scaling and spindle morphometric variation.

Sequential Turnovers of Sex Chromosomes in African Clawed Frogs (Xenopus) Suggest Some Genomic Regions Are Good at Sex Determination.

Sexual differentiation is fundamentally important for reproduction, yet the genetic triggers of this developmental process can vary, even between closely related species. Recent studies have uncovered, for example, variation in the genetic triggers for sexual differentiation within and between species of African clawed frogs (genus Xenopus). Here, we extend these discoveries by demonstrating that yet another sex determination system exists in Xenopus, specifically in the species Xenopus borealis. This system evolved recently in an ancestor of X. borealis that had the same sex determination system as X. laevis, a system which itself is newly evolved. Strikingly, the genomic region carrying the sex determination factor in X. borealis is homologous to that of therian mammals, including humans. Our results offer insights into how the genetic underpinnings of conserved phenotypes evolve, and suggest an important role for cooption of genetic building blocks with conserved developmental roles.

Nucleolar Dominance and Repression of 45S Ribosomal RNA Genes in Hybrids between Xenopus borealis and X. muelleri (2n = 36)

Nucleolar dominance is a dramatic disruption in the formation of nucleoli and the expression of ribosomal RNA (rRNA) genes, characteristic of some plant and animal hybrids. Here, we report that F₁ hybrids produced from reciprocal crosses between 2 sister species of Xenopus clawed frogs, X. muelleri and X. borealis, undergo nucleolar dominance somewhat distinct from a pattern previously reported in hybrids between phylogenetically more distant Xenopus species. Patterns of nucleolar development, 45S rRNA expression, and gene copy inheritance were investigated using a combination of immunostaining, pyrosequencing, droplet digital PCR, flow cytometry, and epigenetic inhibition. In X. muelleri × X. borealis hybrids, typically only 1 nucleolus is formed, and 45S rRNA genes are predominantly expressed from 1 progenitor's alleles, X. muelleri, regardless of the cross-direction. These changes are accompanied by an extensive (∼80%) loss of rRNA gene copies in the hybrids relative to their parents, with the transcriptionally underdominant variant (X. borealis) being preferentially lost. Chemical treatment of hybrid larvae with a histone deacetylase inhibitor resulted in a partial derepression of the underdominant variant. Together, these observations shed light on the genetic and epigenetic basis of nucleolar dominance as an underappreciated manifestation of genetic conflicts within a hybrid genome.

Xenopus males lose characteristics through different strategies

![Figure][1] Female (left) and male Xenopus borealis . Photo credit: Elizabeth Leininger. Some males really go to town when putting on a show for the ladies. Male peacocks flounce around with elaborate feather trains while male dear strut their stuff with impressive antler racks. However

A comparison of host-defense peptides in skin secretions of female Xenopus laevis × Xenopus borealis and X. borealis × X. laevis F1 hybrids

Peptidomic analysis was used to compare the diversity of host-defense peptides in norepinephrine-stimulated skin secretions from laboratory-generated female F1 hybrids of Xenopus laevis and Xenopus borealis (Pipidae). Skin secretions of hybrids with maternal X. laevis (XLB) contained 12 antimicrobial peptides (AMPs), comprising 8 from X. laevis and 4 from X. borealis. Magainin-B1, XPF-B1, PGLa-B1 CPF-B2, CPF-B3 and CPF-B4 from X. borealis and XPF-1, XPF-2, and CPF-6 from X. laevis were not detected and CPF-1 and CPF-7 were present in low concentration. The secretions contained caerulein and caerulein-B1 derived from both parents but lacked X. laevis xenopsin and X. borealis caerulein-B2. Skin secretions of hybrids with maternal X. borealis (XBL) contained 14 AMPs comprising 6 from X. borealis and 8 from X. laevis. Magainin-B1, XPF-B1, PGLa-B1, CPF-B2, XPF-1, CPF-5, and CPF-7 were absent and CPF-B3, CPF-B4, CPF-1 and CPF-6 were present only in low concentration. Xenopsin and caerulein were identified in the secretions but caerulein-B2 was absent and caerulein-B1was present in low concentration. No peptides were identified in secretions of either XLB or XBL hybrids that were not present in the parental species. The data indicate that hybridization between X. laevis and X. borealis results in increased diversity of host-defense peptides in skin secretions but point to extensive AMP gene silencing compared with previously studied female X. laevis×X. muelleri F1 hybrids and no novel peptide expression.

Selective association between nucleosomes with identical DNA sequences

Self-assembly is the autonomous organization of constituents into higher order structures or assemblages and is a fundamental mechanism in biological systems. There has been an unfounded idea that self-assembly may be used in the sensing and pairing of homologous chromosomes or chromatin, including meiotic chromosome pairing, polytene chromosome formation in Diptera and transvection. Recent studies proved that double-stranded DNA molecules have a sequence-sensing property and can self-assemble, which may play a role in the above phenomena. However, to explain these processes in terms of self-assembly, it first must be proved that nucleosomes retain a DNA sequence-sensing property and can self-assemble. Here, using atomic force microscopy (AFM)-based analyses and a quantitative interaction assay, we show that nucleosomes with identical DNA sequences preferentially associate with each other in the presence of Mg2+ ions. Using Xenopus borealis 5S rDNA nucleosome-positioning sequence and 601 and 603 sequences, homomeric or heteromeric octa- or tetranucleosomes were reconstituted in vitro and induced to form weak intracondensates by MgCl2. AFM clearly showed that DNA sequence-based selective association occurs between nucleosomes with identical DNA sequences. Selective association was also detected between mononucleosomes. We propose that nucleosome self-assembly and DNA self-assembly constitute the mechanism underlying sensing and pairing of homologous chromosomes or chromatin.

Next generation sequencing and comparative analyses of Xenopus mitogenomes

BackgroundMitochondrial genomes comprise a small but critical component of the total DNA in eukaryotic organisms. They encode several key proteins for the cell’s major energy producing apparatus, the mitochondrial respiratory chain. Additonally, their nucleotide and amino acid sequences are of great utility as markers for systematics, molecular ecology and forensics. Their characterization through nucleotide sequencing is a fundamental starting point in mitogenomics. Methods to amplify complete mitochondrial genomes rapidly and efficiently from microgram quantities of tissue of single individuals are, however, not always available. Here we validate two approaches, which combine long-PCR with Roche 454 pyrosequencing technology, to obtain two complete mitochondrial genomes from individual amphibian species.ResultsWe obtained two new xenopus frogs (Xenopus borealis and X. victorianus) complete mitochondrial genome sequences by means of long-PCR followed by 454 of individual genomes (approach 1) or of multiple pooled genomes (approach 2), the mean depth of coverage per nucleotide was 9823 and 186, respectively. We also characterised and compared the new mitogenomes against their sister taxa; X. laevis and Silurana tropicalis, two of the most intensely studied amphibians. Our results demonstrate how our approaches can be used to obtain complete amphibian mitogenomes with depths of coverage that far surpass traditional primer-walking strategies, at either the same cost or less. Our results also demonstrate: that the size, gene content and order are the same among xenopus mitogenomes and that S. tropicalis form a separate clade to the other xenopus, among which X. laevis and X. victorianus were most closely related. Nucleotide and amino acid diversity was found to vary across the xenopus mitogenomes, with the greatest diversity observed in the Complex 1 gene nad4l and the least diversity observed in Complex 4 genes (cox1-3). All protein-coding genes were shown to be under strong negative (purifying selection), with genes under the strongest pressure (Complex 4) also being the most highly expressed, highlighting their potentially crucial functions in the mitochondrial respiratory chain.ConclusionsNext generation sequencing of long-PCR amplicons using single taxon or multi-taxon approaches enabled two new species of Xenopus mtDNA to be fully characterized. We anticipate our complete mitochondrial genome amplification methods to be applicable to other amphibians, helpful for identifying the most appropriate markers for differentiating species, populations and resolving phylogenies, a pressing need since amphibians are undergoing drastic global decline. Our mtDNAs also provide templates for conserved primer design and the assembly of RNA and DNA reads following high throughput “omic” techniques such as RNA- and ChIP-seq. These could help us better understand how processes such mitochondrial replication and gene expression influence xenopus growth and development, as well as how they evolved and are regulated.

Host-defense peptides in skin secretions of African clawed frogs (Xenopodinae, Pipidae)

African clawed frogs of the Xenopodinae (Xenopus+Silurana) constitute a well-defined system in which to study the evolutionary trajectory of duplicated genes and are a source of antimicrobial peptides with therapeutic potential. Allopolyploidization events within the Xenopodinae have given rise to tetraploid, octoploid, and dodecaploid species. The primary structures and distributions of host-defense peptides from the tetraploid frogs Xenopus borealis, Xenopus clivii, Xenopus laevis, Xenopus muelleri, “X. muelleri West”, and Xenopus petersii may be compared with those from the octoploid frogs Xenopus amieti and X. andrei. Similarly, components in skin secretions from the diploid frog Silurana tropicalis may be compared with those from the tetraploid frog Silurana paratropicalis. All Xenopus antimicrobial peptides may be classified in the magainin, peptide glycine-leucine-amide (PGLa), caerulein-precursor fragment (CPF), and xenopsin-precursor fragment (XPF) families. However, the numbers of paralogs from the octoploid frogs were not significantly greater than the corresponding numbers from the tetraploid frogs. Magainins were not identified in skin secretions of Silurana frogs and the multiplicity of the PGLa, CPF, and XPF peptides from S. paratropicalis was not greater than that of S. tropicalis. The data indicate, therefore, that nonfunctionalization (gene silencing) has been the most common fate of antimicrobial peptide genes following polyploidization. While some duplicated gene products retain high antimicrobial potency (subfunctionalization), the very low activity of others suggests that they may be evolving towards a new biological role (neofunctionalization). CPF-AM1 and PGLa-AM1 from X. amieti show potential for development into anti-infective agents for use against antibiotic-resistant Gram-negative bacteria.

Caerulein-and xenopsin-related peptides with insulin-releasing activities from skin secretions of the clawed frogs, Xenopus borealis and Xenopus amieti (Pipidae)

Caerulein-related peptides were identified in norepinephrine-stimulated skin secretions of the tetraploid frog Xenopus borealis and the octoploid frog Xenopus amieti using negative ion electrospray mass spectrometry and their primary structures determined by positive ion tandem (MS/MS) mass spectrometry. X. borealis caerulein-B1 (pGlu-Gln-Asp-Tyr(SO 3)-Gly-Thr-Gly-Trp-Met-Asp-Phe.NH2) contains an additional Gly 5 residue compared with X. laevis caerulein and caerulein-B2 (pGlu-Asp-Tyr(SO 3)-Thr-Gly-Trp-Met-Asp-Phe.NH2) contains a Gln 2 deletion. X. amieti caerulein was identical to the X. laevis peptide. In addition, xenopsin, identical to the peptide from X. laevis, together with xenopsin-AM2 (pGlu-Gly-Arg-Arg-Pro-Trp-Ile- Leu) that contains the substitution Lys 3 → Arg were isolated from X. amieti secretions. X. borealis caerulein-B1, and X. amieti xenopsin and xenopsin-AM2 produced significant ( P < 0.05) and concentration-dependent stimulations of insulin release from the rat BRIN-BD11 clonal β cell line at concentrations ⩾30 nM. The peptides did not stimulate the release of lactate dehydrogenase at concentrations up to 3 μM demonstrating that the integrity of the plasma membrane had been preserved. While their precise biological role is unclear, the caerulein- and xenopsin-related peptides may constitute a component of the animal’s chemical defenses against predators.

Structure determination by MALDI-IRMPD mass spectrometry and exoglycosidase digestions of O-linked oligosaccharides from Xenopus borealis egg jelly

Differences in the fertilization behavior of Xenopus borealis from X. laevis and X. tropicalis suggest differences in the glycosylation of the egg jellies. To test this assumption, O-linked glycans were chemically released from the egg jelly coat glycoproteins of X. borealis. Over 50 major neutral glycans were observed, and no anionic glycans were detected from the released O-glycan pool. Preliminary structures of ∼30 neutral oligosaccharides were determined using matrix-assisted laser desorption/ionization (MALDI) infrared multiphoton dissociation tandem mass spectrometry (MS). The mass fingerprint of a group of peaks for the core-2 structure of O-glycans was conserved in the tandem mass spectra and was instrumental in rapid and efficient structure determination. Among the 29 O-glycans, 22 glycans contain the typical core-2 structure, 3 glycans have the core-1 structure and 2 glycans contained a previously unobserved core structure with hexose at the reducing end. There were seven pairs of structural isomers observed in the major O-linked oligosaccharides. To further elucidate the structures of a dozen O-linked glycans, specific and targeted exoglycosidase digestions were carried out and the products were monitored with MALDI-MS. Reported here are the elucidated structures of O-linked oligosaccharides from glycoproteins of X. borealis egg jelly coats. The structural differences in O-glycans from jelly coats of X. borealis and its close relatives may provide a better understanding of the structure-function relationships and the role of glycans in the fertilization process within Xenopodinae.

Antimicrobial peptides with therapeutic potential from skin secretions of the Marsabit clawed frog Xenopus borealis (Pipidae)

Nine peptides with differential growth inhibitory activity against Escherichia coli and Staphylococcus aureus were isolated from norepinephrine-stimulated skin secretions of the tetraploid frog Xenopus borealis Parker, 1936 (Pipidae). Structural characterization of the peptides demonstrated that they were orthologous to magainin-2 (1 peptide), peptide glycine–leucine-amide, PGLa (2 peptides), caerulein-precursor fragments, CPF (4 peptides), and xenopsin-precursor fragments, XPF (2 peptides), previously isolated from Xenopus laevis and X. amieti. In addition, a second magainin-related peptide (G**KFLHSAGKFGKAFLGEVMIG) containing a two amino acid residue deletion compared with magainin-2 was identified that had only weak antimicrobial activity. The peptide with the greatest potential for development into a therapeutically valuable anti-infective agent was CPF-B1 (GLGSLLGKAFKIGLKTVGKMMGGAPREQ) with MIC = 5 μM against E. coli, MIC = 5 μM against S. aureus, and MIC = 25 μM against Candida albicans, and low hemolytic activity against human erythrocytes (LC 50 > 200 μM). This peptide was also the most abundant antimicrobial peptide in the skin secretions. CPF-B1 was active against clinical isolates of the nosocomial pathogens, methicillin-resistant S. aureus (MRSA) and multidrug-resistant Acinetobacter baumannii (MDRAB) with MIC values in the range 4–8 μM.

Diet composition ofXenopus borealisin Taita Hills: effects of habitat and predator size

AbstractFrogs in the genusXenopusare ubiquitous in sub‐Saharan Africa, yet very little is recorded on their ecology. They are commonly found in anthropogenically disturbed habitats, but how do these compare to conspecifics from natural habitats? The diet ofXenopus borealisfrom three different sites in Taita Hills, Kenya was established based on a sample of 77 (54 females and 23 males) specimens from two disturbed and one pristine sites.Xenopus borealisfrom all the sites was found to be a dietary generalist, feeding predominantly on invertebrates. A total of twelve invertebrate orders both terrestrial and aquatic were recorded in addition to amphibian eggs, tadpoles and fish. Frogs from the pristine forest were smaller and had ingested more terrestrial prey items than frogs in the disturbed open habitat ponds. The stomach content (both by mass and quantity) was independent of body size. The results suggest thatX. borealisis an opportunistic generalist predator which may be constrained by food availability in its natural habitat. However, disturbed habitats provide abundant food items which are enough to significantly increase the mean size of the population.

Evolution of the Xenopus piggyBac Transposon Family TxpB: Domesticated and Untamed Strategies of Transposon Subfamilies

A new family, termed TxpB, of DNA transposons belonging to the piggyBac superfamily was found in 3 Xenopus species (Xenopus tropicalis, Xenopus laevis, and Xenopus borealis). Two TxpB subfamilies of Kobuta and Uribo1 were found in all the 3 species, and another subfamily termed Uribo2 was found in X. tropicalis. Molecular phylogenetic analyses of their open reading frames (ORFs) revealed that TxpB transposons have been maintained for over 100 Myr. Both the Uribo1 and the Uribo2 ORFs were present as multiple copies in each genome, and some of them were framed by terminal inverted repeat sequences. In contrast, all the Kobuta ORFs were present as a single copy in each genome and exhibited high evolutionary conservation, suggesting domestication of Kobuta genes by the host. Genomic insertion polymorphisms of the Uribo1 and Uribo2 transposons (nonautonomous type) were observed in a single species of X. tropicalis, indicating recent transposition events. Transfection experiments in cell culture revealed that an expression vector construct for the intact Uribo2 ORF caused precise excision of a nonautonomous Uribo2 element from the target vector construct but that for the Kobuta ORF did not. The present results support our viewpoint that some Uribo2 members are naturally active autonomous transposons, whereas Kobuta members may be domesticated by hosts.

Overexpression of repulsive guidance molecule (RGM) a induces cell death through Neogenin in early vertebrate development

Repulsive guidance molecule (RGM) a is a glycosylphosphatidylinositol (GPI)-anchored plasma membrane protein that has been implicated in chemorepulsive axon guidance. Although RGMa binds the transmembrane receptor Neogenin, the developmental events controlled by the RGMa-Neogenin interactions in vivo remain largely unknown. We have cloned full-length RGMa from Xenopus borealis for the first time and identified two homologous genes referred to as RGMa1 and RGMa2. Here we show RGMa1 overexpression at 2-cell-stage resulted in cell death, which lead to an early embryonic lethal phenotype of the embryos. Time-lapse photomicroscopy revealed that embryos began to show initial morphological defects from approximately 5 h post-fertilization (hpf) which was then followed by extensive blastomere cell death at approximately 11 hpf. This phenotype was rescued by simultaneous knock down of RGMa using translation blocking anti-sense morpholinos. Knock down of the RGMa1 receptor Neogenin in RGMa1 overexpressing embryos was also able to rescue the phenotype. Together these results indicated that RGMa1 was signalling through Neogenin to induce cell death in the early embryo. While previous studies have suggested that Neogenin is a dependence receptor that induces cell death in the absence of RGM, we have instead shown that Neogenin-RGM interactions induce cell death in the early embryo. The roles of RGMa1 and Neogenin appear to be context specific so that their co-ordinated and regulated expressions are essential for normal development of the vertebrate embryo.

The Core Histone Tail Domains Contribute to Sequence-dependent Nucleosome Positioning

The precise positioning of nucleosomes plays a critical role in the regulation of gene expression by modulating the DNA binding activity of trans-acting factors. However, molecular determinants responsible for positioning are not well understood. We examined whether the removal of the core histone tail domains from nucleosomes reconstituted with specific DNA fragments led to alteration of translational positions. Remarkably, we find that removal of tail domains from a nucleosome assembled on a DNA fragment containing a Xenopus borealis somatic-type 5S RNA gene results in repositioning of nucleosomes along the DNA, including two related major translational positions that move about 20 bp further upstream with respect to the 5S gene. In a nucleosome reconstituted with a DNA fragment containing the promoter of a Drosophila alcohol dehydrogenase gene, several translational positions shifted by about 10 bp along the DNA upon tail removal. However, the positions of nucleosomes assembled with a DNA fragment known to have one of the highest binding affinities for core histone proteins in the mouse genome were not altered by removal of core histone tail domains. Our data support the notion that the basic tail domains bind to nucleosomal DNA and influence the selection of the translational position of nucleosomes and that once tails are removed movement between translational positions occurs in a facile manner on some sequences. However, the effect of the N-terminal tails on the positioning and movement of a nucleosome appears to be dependent on the DNA sequence such that the contribution of the tails can be masked by very high affinity DNA sequences. Our results suggest a mechanism whereby sequence-dependent nucleosome positioning can be specifically altered by regulated changes in histone tail-DNA interactions in chromatin.

Experimental analysis of lens-forming capacity inXenopus borealis larvae

Previously, the only anuran amphibians known to have the capacity to regenerate a lens after lentectomy were Xenopus laevis and Xenopus tropicalis. This regeneration process occurs during the larval life through transdifferentiation of the outer cornea promoted by inductive factors produced by the retina and accumulated inside the vitreous chamber. However, the capacity of X. tropicalis to regenerate a lens is much lower than that of X. laevis. This study demonstrates that Xenopus borealis, a species more closely related to X. laevis than to X. tropicalis, is not able to regenerate a lens after lentectomy. Nevertheless, some morphological modifications corresponding to the first stages of lens regeneration in X. laevis were observed in the outer cornea of X. borealis. This suggested that in X borealis the regeneration process was blocked at early stages. Results from histological analysis of X. borealis and X. laevis lentectomized eyes and from implantation of outer cornea fragments into the vitreous and anterior chambers demonstrated that: (i) in X. borealis eye, the lens-forming competence in the outer cornea and inductive factors in the vitreous chamber are both present, (ii) no inhibiting factors are present in the anterior chamber, the environment where lens regeneration begins, (iii) the inability of X. borealis to regenerate a lens after lentectomy is due to an inhibiting action exerted by the inner cornea on the spreading of the retinal factor from the vitreous chamber towards the outer cornea. This mechanical inhibition is assured by two distinctive features of X. borealis eye in comparison with X. laevis eye: (i) a weaker and slower response to the retinal inducer by the outer cornea; (ii) a stronger and faster healing of the inner cornea. Unlike X. tropicalis and similar to X. laevis, in X. borealis the competence to respond to the retinal factor is not restricted to the corneal epithelium but also extends to the pericorneal epidermis.

Accommodation and Repair of a UV Photoproduct in DNA at Different Rotational Settings on the Nucleosome Surface

Cyclobutane-thymine dimers (CTDs), the most common DNA lesion induced by UV radiation, cause 30 degrees bending and 9 degrees unwinding of the DNA helix. We prepared site-specific CTDs within a short sequence bracketed by strong nucleosome-positioning sequences. The rotational setting of CTDs over one turn of the helix near the dyad center on the histone surface was analyzed by hydroxyl radical footprinting. Surprisingly, the position of CTDs over one turn of the helix does not affect the rotational setting of DNA on the nucleosome surface. Gel-shift analysis indicates that one CTD destabilizes histone-DNA interactions by 0.6 or 1.1 kJ/mol when facing away or toward the histone surface, respectively. Thus, 0.5 kJ/mol energy penalty for a buried CTD is not enough to change the rotational setting of sequences with strong rotational preference. The effect of rotational setting on CTD removal by nucleotide excision repair (NER) was examined using Xenopus oocyte nuclear extracts. The NER rates are only 2-3 times lower in nucleosomes and change by only 1.5-fold when CTDs face away or toward the histone surface. Therefore, in Xenopus nuclear extracts, the rotational orientation of CTDs on nucleosomes has surprisingly little effect on rates of repair. These results indicate that nucleosome dynamics and/or chromatin remodeling may facilitate NER in gaining access to DNA damage in nucleosomes.

Conformation of Reconstituted Mononucleosomes and Effect of Linker Histone H1 Binding Studied by Scanning Force Microscopy

The conformation of mononucleosome complexes reconstituted with recombinant core histones on a 614-basepair-long DNA fragment containing the Xenopus borealis 5S rRNA nucleosome positioning sequence was studied by scanning/atomic force microscopy in the absence or presence of linker histone H1. Imaging without prior fixation was conducted with air-dried samples and with mononucleosomes that were injected directly into the scanning force microscopy fluid cell and visualized in buffer. From a quantitative analysis of ∼1,700 complexes, the following results were obtained: i), In the absence of H1, a preferred location of the nucleosome at the X. borealis 5S rRNA sequence in the center of the DNA was detected. From the distribution of nucleosome positions, an energy difference of binding to the 5S rRNA sequence of ΔΔ G ≈ 3 kcal mol −1 as compared to a random sequence was estimated. Upon addition of H1, a significantly reduced preference of nucleosome binding to this sequence was observed. ii), The measured entry-exit angles of the DNA at the nucleosome in the absence of H1 showed two maxima at 81 ± 29° and 136 ± 18° (air-dried samples), and 78 ± 25° and 137 ± 25° (samples imaged in buffer solution). In the presence of H1, the species with the smaller entry-exit angle was stabilized, yielding average values of 88 ± 34° for complexes in air and 85 ± 10° in buffer solution. iii), The apparent contour length of the nucleosome complexes was shortened by 34 ± 13 nm as compared to the free DNA due to wrapping of the DNA around the histone octamer complex. Considering an 11 nm diameter of the nucleosome core complex, this corresponds to a total of 145 ± 34 basepairs that are wound around the nucleosome.