LINE L1 retrotransposable element is targeted during the initial stages of apoptotic DNA fragmentation

Using a directional cloning strategy, DNA sequence information was obtained corresponding to the site of early radiation-induced apoptotic DNA fragmentation within the human lymphoblastoid cell line TK6. Data were obtained from 88 distinct clones comprising approximately 65 kbp of sequenced material. Analysis of all cloned material showed that sequences in the 10 bp immediately adjacent to the cleavage sites were enriched in short oligoT tracts. The proportion of repetitive DNA within the entire cloned material was found to be within the normal range. However the distribution of Alu and LINE repetitive DNA were biased to positions at or adjacent to the apoptotic cleavage site. In particular, a non-random distribution of five cleavage sites was found clustered within the second ORF of the LINE L1 that partially overlapped with two binding sites for the nuclear matrix-associated protein SATB1. Three other clones, containing alpha satellite elements, were also linked to a DNA matrix binding function. These data indicate that the site of chromatin loop formation at the nuclear matrix may be a specific target for early DNA fragmentation events during apoptosis.     

Tetracycline promoter mutations decrease non-B DNA structural transitions, negative linking differences and deletions in recombinant plasmids in Escherichia coli

The ability to clone a variety of sequences with varying capabilities of adopting non-B structures (left-handed Z-DNA, cruciforms or triplexes) into three loci of pBR322 was investigated. In general, the inserts were stable (non-deleted) in the EcoRI site (an untranslated region) of pBR322. However, sequences most likely to adopt left-handed Z-DNA or triplexes in vivo suffered deletions when cloned into the BamHI site, which is located in the tetracycline resistance structural gene (tet). Conversely, when the promoter for the tet gene was altered by filling-in the unique HindIII or ClaI sites, the inserts in the BamHI site were not deleted. Concomitantly, the negative linking differences of the plasmids were reduced. Also, inserts with a high potential to adopt Z-DNA conformations were substantially deleted in the PvuII site of pBR322 (near the replication origin and the copy number control region), but were less deleted if the tet promoter was insertion-mutated. The deletion phenomena are due to the capacity of these sequences to adopt left-handed Z-DNA or triplexes in vivo since shorter inserts, less prone to form non-B DNA structures, or random sequences, did not exhibit this behavior. Sequences with the potential to adopt cruciforms were stable in all sites under all conditions. These results reveal a complex interrelationship between insert deletions (apparently the result of genetic recombination), negative supercoiling, and the formation of non-B DNA structures in living Escherichia coli cells.     

Potential non-B DNA regions in the human genome are associated with higher rates of nucleotide mutation and expression variation

While individual non-B DNA structures have been shown to impact gene expression, their broad regulatory role remains elusive. We utilized genomic variants and expression quantitative trait loci (eQTL) data to analyze genome-wide variation propensities of potential non-B DNA regions and their relation to gene expression. Independent of genomic location, these regions were enriched in nucleotide variants. Our results are consistent with previously observed mutagenic properties of these regions and counter a previous study concluding that G-quadruplex regions have a reduced frequency of variants. While such mutagenicity might undermine functionality of these elements, we identified in potential non-B DNA regions a signature of negative selection. Yet, we found a depletion of eQTL-associated variants in potential non-B DNA regions, opposite to what might be expected from their proposed regulatory role. However, we also observed that genes downstream of potential non-B DNA regions showed higher expression variation between individuals. This coupling between mutagenicity and tolerance for expression variability of downstream genes may be a result of evolutionary adaptation, which allows reconciling mutagenicity of non-B DNA structures with their location in functionally important regions and their potential regulatory role.     

Double-strand break formation by the RAG complex at the bcl-2 major breakpoint region and at other non-B DNA structures in vitro

The most common chromosomal translocation in cancer, t(14;18) at the 150-bp bcl-2 major breakpoint region (Mbr), occurs in follicular lymphomas. The bcl-2 Mbr assumes a non-B DNA conformation, thus explaining its distinctive fragility. This non-B DNA structure is a target of the RAG complex in vivo, but not because of its primary sequence. Here we report that the RAG complex generates at least two independent nicks that lead to double-strand breaks in vitro, and this requires the non-B DNA structure at the bcl-2 Mbr. A 3-bp mutation is capable of abolishing the non-B structure formation and the double-strand breaks. The observations on the bcl-2 Mbr reflect more general properties of the RAG complex, which can bind and nick at duplex-single-strand transitions of other non-B DNA structures, resulting in double-strand breaks in vitro. Hence, the present study reveals novel insight into a third mechanism of action of RAGs on DNA, besides the standard heptamer/nonamer-mediated cleavage in V(D)J recombination and the in vitro transposase activity.     

Premutation huntingtin allele adopts a non-B conformation and contains a hot spot for DNA damage

The expansion of a CAG trinucleotide repeat (TNR) sequence has been linked to several neurological disorders, for example, Huntington's disease (HD). In HD, healthy individuals have 5-35 CAG repeats. Those with 36-39 repeats have the premutation allele, which is known to be prone to expansion. In the disease state, greater than 40 repeats are present. Interestingly, the formation of non-B DNA conformations by the TNR sequence is proposed to contribute to the expansion. Here we provide the first structural and thermodynamic analysis of a premutation length TNR sequence. Using chemical probes of nucleobase accessibility, we found that similar to (CAG)(10), the premutation length sequence (CAG)(36) forms a stem-loop hairpin and contains a hot spot for DNA damage. Additionally, calorimetric analysis of a series of (CAG)(n) sequences, that includes repeat tracts in both the healthy and premutation ranges, reveal that thermodynamic stability increases linearly with the number of repeats. Based on these data, we propose that while non-B conformations can be formed by TNR tracts found in both the healthy and premutation allele, only sequences containing at least 36 repeats have sufficient thermodynamic stability to contribute to expansion.     

Associations between intronic non-B DNA structures and exon skipping

Non-B DNA structures are abundant in the genome and are often associated with critical biological processes, including gene regulation, chromosome rearrangement and genome stabilization. In particular, G-quadruplex (G4) may affect alternative splicing based on its ability to impede the activity of RNA polymerase II. However, the specific role of non-B DNA structures in splicing regulation still awaits investigation. Here, we provide a genome-wide and cross-species investigation of the associations between five non-B DNA structures and exon skipping. Our results indicate a statistically significant correlation of each examined non-B DNA structures with exon skipping in both human and mouse. We further show that the contributions of non-B DNA structures to exon skipping are influenced by the occurring region. These correlations and contributions are also significantly different in human and mouse. Finally, we detailed the effects of G4 by showing that occurring on the template strand and the length of G-run, which is highly related to the stability of a G4 structure, are significantly correlated with exon skipping activity. We thus show that, in addition to the well-known effects of RNA and protein structure, the relative positional arrangement of intronic non-B DNA structures may also impact exon skipping.     

Mitochondrial DNA deletions are associated with non-B DNA conformations

Mitochondrial DNA (mtDNA) deletions are a primary cause of mitochondrial disease and are believed to contribute to the aging process and to various neurodegenerative diseases. Despite strong observational and experimental evidence, the molecular basis of the deletion process remains obscure. In this study, we test the hypothesis that the primary cause of mtDNA vulnerability to breakage resides in the formation of non-B DNA conformations, namely hairpin, cruciform and cloverleaf-like elements. Using the largest database of human mtDNA deletions built thus far (753 different cases), we show that site-specific breakage hotspots exist in the mtDNA. Furthermore, we discover that the most frequent deletion breakpoints occur within or near predicted structures, a result that is supported by data from transgenic mice with mitochondrial disease. There is also a significant association between the folding energy of an mtDNA region and the number of breakpoints that it harbours. In particular, two clusters of hairpins (near the D-loop 3'-terminus and the L-strand origin of replication) are hotspots for mtDNA breakage. Consistent with our hypothesis, the highest number of 5'- and 3'-breakpoints per base is found in the highly structured tRNA genes. Overall, the data presented in this study suggest that non-B DNA conformations are a key element of the mtDNA deletion process.     

Evolutionary diversity and potential recombinogenic role of integration targets of Non-LTR retrotransposons

Short interspersed elements (SINEs) make up a significant fraction of total DNA in mammalian genomes, providing a rich substrate for chromosomal rearrangements by SINE-SINE recombinations. Proliferation of mammalian SINEs is mediated primarily by long interspersed element 1 (L1) non-long terminal repeat retrotransposons that preferentially integrate at DNA sequence targets with an average length of approximately 15 bp and containing conserved endonucleolytic nicking signals at both ends. We report that sequence variations in the first of the two nicking signals, represented by a 5'-TT-AAAA consensus sequence, affect the position of the second signal thus leading to target site duplications (TSDs) of different lengths. The length distribution of TSDs appears to be affected also by L1-encoded enzyme variants because targets with the same 5' nicking site can be of different average lengths in different mammalian species. Taking this into account, we reanalyzed the second nicking site and found that it is larger and includes more conserved sites than previously appreciated, with a consensus of 5'-ANTNTN-AA. We also studied potential involvement of the nicking sites in stimulating recombinations between SINEs. We determined that SINEs retaining TSDs with perfect 5'-TT-AAAA nicking sites appear to be lost relatively rapidly from the human and rat genomes and less rapidly from dog. We speculate that the introduction of DNA breaks induced by recurring endonucleolytic attacks at these sites, combined with the ubiquitousness of SINEs, may significantly promote recombination between repetitive elements, leading to the observed losses. At the same time, new L1 subfamilies may be selected for "incompatibility" with preexisting targets. This provides a possible driving force for the continual emergence of new L1 subfamilies which, in turn, may affect selection of L1-dependent SINE subfamilies.     

Common physical properties of DNA affecting target site selection of sleeping beauty and other Tc1/mariner transposable elements

Sleeping Beauty (SB) is the most active Tc1/mariner-type transposable element in vertebrates, and is therefore a valuable vector for transposon mutagenesis in vertebrate models and for human gene therapy. We have analyzed factors affecting target site selection of SB in mammalian cells, by generating transposition events from extrachromosomal plasmids to chromosomes. In contrast to the local hopping observed when transposition is induced from a chromosomal context, mapping of 138 unique SB insertions on human chromosomes showed a fairly random genomic distribution, and a 35% occurrence of transposition into genes. Inspection of the DNA flanking the sites of element integration revealed significant differences from random DNA in both primary sequence and physical properties. The consensus sequence of SB target sites was found to be a palindromic AT-repeat, ATATATAT, in which the central TA is the canonical target site. We found however, that target site selection is determined primarily on the level of DNA structure, and not by specific base-pair interactions. Computational analyses revealed that insertion sites tend to have a bendable structure and a palindromic pattern of potential hydrogen-bonding sites in the major groove of the DNA. These features appear conserved in the Tc1/mariner family of transposons and in other, distantly related elements that share a common catalytic domain of the transposase, and integrate fairly randomly. No similar target site preference was found for non-randomly integrating elements. Our results suggest common factors influencing target site selection of a wide range of transposable elements.     

Co-amplification of L1 line elements with localised low copy repeats in Giemsa dark bands: implications for genome organisation.

A repeat sequence island, located at the A3 Giemsa dark band on the mouse X chromosome and consisting of 50 copies of a localised long complex repeat unit (LCRU), features an unusually high concentration of L1 LINE repeat sequences juxtaposed and inserted within the LCRU. Sequence analysis of three independent genomic clones containing L1 LINE elements juxtaposed with the LCRU demonstrates a common junction sequence at the L1/LCRU boundary, suggesting that the high concentration of L1 LINE sequences in the repeat sequence island has arisen by association of an L1 element with an LCRU followed by amplification. The LCRU target site at this common junction sequence bears no resemblance to the target site of an L1 element inserted within one LCRU, indicating there is no specific preferential target site for L1 integration. We propose that co-amplification of L1 LINE elements with localised low copy repeat families throughout the genome could have a major effect on the chromosomal distribution of L1 LINE elements.     

Human transposon-like elements insert at a preferred target site: evidence for a retrovirally mediated process.

Members of the human transposon-like family of repetitive sequences (called THE 1 repeats) like many other repetitive DNA sequences are flanked by short direct repeats. Comparison of the base sequences of twelve examples of these flanking direct repeats indicates that THE 1 repeats insert into a preferred genomic target site. In one case, we have identified the sequence of an empty site into which a THE 1 element inserted. The sequence of this empty site and sequences of truncated THE 1 LTRs are consistent with a retroviral mechanism for the insertion of THE 1 elements. Truncated transposon structures illustrate for the first time that intermediate structures of retrotransposition may also be integrated into the genome.     

Increased negative superhelical density in vivo enhances the genetic instability of triplet repeat sequences

The influence of negative superhelical density on the genetic instabilities of long GAA.TTC, CGG.CCG, and CTG.CAG repeat sequences was studied in vivo in topologically constrained plasmids in Escherichia coli. These repeat tracts are involved in the etiologies of Friedreich ataxia, fragile X syndrome, and myotonic dystrophy type 1, respectively. The capacity of these DNA tracts to undergo deletions-expansions was explored with three genetic-biochemical approaches including first, the utilization of topoisomerase I and/or DNA gyrase mutants, second, the specific inhibition of DNA gyrase by novobiocin, and third, the genetic removal of the HU protein, thus lowering the negative supercoil density (-sigma). All three strategies revealed that higher -sigma in vivo enhanced the formation of deleted repeat sequences. The effects were most pronounced for the Friedreich ataxia and the fragile X triplet repeat sequences. Higher levels of -sigma stabilize non-B DNA conformations (i.e. triplexes, sticky DNA, flexible and writhed DNA, slipped structures) at appropriate repeat tracts; also, numerous prior genetic instability investigations invoke a role for these structures in promoting the slippage of the DNA complementary strands. Thus, we propose that the in vivo modulation of the DNA structure, localized to the repeat tracts, is responsible for these behaviors. Presuming that these interrelationships are also found in humans, dynamic alterations in the chromosomal nuclear matrix may modulate the -sigma of certain DNA regions and, thus, stabilize/destabilize certain non-B conformations which regulate the genetic expansions-deletions responsible for the diseases.     

Stability and strand asymmetry in the non-B DNA structure at the bcl-2 major breakpoint region

The t(14;18) translocation involving the Ig heavy chain locus and the BCL-2 gene is the single most common chromosomal translocation in human cancer. Recently we reported in vitro and in vivo chemical probing data indicating that the 150-bp major breakpoint region (Mbr), which contains three breakage subregions (hotspots) (known as peaks I, II, and III), has single-stranded character and hence a non-B DNA conformation. Although we could document the non-B DNA structure formation at the bcl-2 Mbr, the structural studies were limited to chemical probing. Therefore, in the present study, we used multiple methods including circular dichroism to detect the non-B DNA at the bcl-2 Mbr. We established a new gel shift method to detect the altered structure at neutral pH on shorter DNA fragments containing the bcl-2 Mbr and analyzed the fine structural features. We found that the single-stranded region in the non-B DNA structure observed is stable for days and is asymmetric with respect to the Watson and Crick strands. It could be detected by oligomer probing, a bisulfite modification assay, or a P1 nuclease assay. We provide evidence that two different non-B conformations exist at peak I in addition to the single one observed at peak III. Finally we used mutagenesis and base analogue incorporation to show that the non-B DNA structure formation requires Hoogsteen pairing. These findings place major constraints on the location and nature of the non-B conformations assumed at peaks I and III of the bcl-2 Mbr.