No abstract

Genomic rearrangements are a frequent source of instability, but the mechanisms involved are poorly understood. A 2.5-kbp poly(purine⅐pyrimidine) sequence from the human PKD1 gene, known to form non-B DNA structures, induced long deletions and other instabilities in plasmids that were mediated by mismatch repair and, in some cases, transcription. The breakpoints occurred at predicted non-B DNA structures. Distance measurements also indicated a significant proximity of alternating purine-pyrimidine and oligo(purine⅐pyrimidine) tracts to breakpoint junctions in 222 gross deletions and translocations, respectively, involved in human diseases. In 11 deletions analyzed, breakpoints were explicable by non-B DNA structure formation. We conclude that alternative DNA conformations trigger genomic rearrangements through recombination-repair activities. G ross chromosomal rearrangements are a common source of genetic instability (1). Thus, characterization of the underlying molecular mechanisms of mutagenesis is fundamental for our understanding of human disease. A hallmark of gross deletions is the presence of short homologous tracts (typically 2-8 bp) at the breakpoints (2), a finding that has prompted speculation as to the two distinct mechanisms postulated to be responsible for their formation. The slipped mispairing model (3) envisages that during lagging strand DNA synthesis, distantly located repeats are brought into close proximity by the looping out of the single-stranded region, thereby enabling the replication complex to ''jump'' from the proximal to the distal repeat and hence bypass the looped structure. Alternative models propose that various types of repetitive sequence elements may serve as substrates for intra-or intermolecular recombination (2, 4). Neither model is satisfactory; slipped mispairing is inconsistent with deletions greater than Ϸ500 bp and deletions manifesting Ͻ4-bp homologies (5-9), whereas the recombination model does not provide a rationale for the initiation of the process.Specific sequence motifs such as direct and inverted repeats, (RY⅐RY) n and (R⅐Y) n , in which R represents purine and Y represents pyrimidine, and four closely spaced G-rich direct repeats [i.e., (G⅐C) 3 ] undergo structural transitions from the orthodox right-handed B-helical duplex to higher energy state non-B structures (slipped hairpin͞loops, cruciforms, left-handed Z-helices, triplexes, and tetraplexes, respectively) (10-12) under torsional stress (negative supercoiling) in vivo.Early articles in bacteria and hamster cells reported isolated cases in which deletions could occur by a recombination-repair reaction mediated by cruciform structures forming at each breakpoint (13,14). Recently, the breakpoint junctions of the human constitutional translocations t(1;22), t(4;22), t(11;22), and t(17;22), which involve a common locus on chromosome 22q11.2, were found to coincide with large (Ͼ95 bp) cruciform structures (15-18), suggesting that this conformation may predispose specific loci to genomic rearrangements.The po...

Circular dichroism and 31P-NMR on synthetic oligomers of (dC-dG) inserted within DNA restriction fragments indicate that the right-handed B-structure can exist in close proximity to the left-handed Z-structure. Also, this salt-induced transition to Z-form in a small (dC-dG) segment (1.3%) of a recombinant plasmid markedly influenced the supercoil of the plasmid. These observations have implications for the postulated role of naturally occurring related simple sequences in the regulation of gene activity.

Microsatellites are abundant in vertebrate genomes, but their sequence representation and length distributions vary greatly within each family of repeats (e.g., tetranucleotides). Biophysical studies of 82 synthetic single-stranded oligonucleotides comprising all tetra-and trinucleotide repeats revealed an inverse correlation between the stability of folded-back hairpin and quadruplex structures and the sequence representation for repeats Ն30 bp in length in nine vertebrate genomes. Alternatively, the predicted energies of base-stacking interactions correlated directly with the longest length distributions in vertebrate genomes. Genome-wide analyses indicated that unstable sequences, such as CAG:CTG and CCG:CGG, were over-represented in coding regions and that micro/minisatellites were recruited in genes involved in transcription and signaling pathways, particularly in the nervous system. Microsatellite instability (MSI) is a hallmark of cancer, and length polymorphism within genes can confer susceptibility to inherited disease. Sequences that manifest the highest MSI values also displayed the strongest base-stacking interactions; analyses of 62 tri-and tetranucleotide repeat-containing genes associated with human genetic disease revealed enrichments similar to those noted for micro/minisatellite-containing genes. We conclude that DNA structure and base-stacking determined the number and length distributions of microsatellite repeats in vertebrate genomes over evolutionary time and that micro/minisatellites have been recruited to participate in both gene and protein function.

Left-handed DNA is shown to exist and elicit a biological response in Escherichia coli. A plasmid encoding the gene for a temperature-sensitive Eco RI methylase (MEco RI) was cotransformed with different plasmids containing inserts that had varying capacities to form left-handed helices or cruciforms with a target Eco RI site in the center or at the ends of the inserts. Inhibition of methylation in vivo was found for the stable inserts with the longest left-handed (presumably Z) helices. In vitro methylation with the purified MEco RI agreed with the results in vivo. Supercoil-induced changes in the structure of the primary helix in vitro provided confirmation that left-handed helices were responsible for this behavior. The presence in vivo of left-handed inserts elicits specific deletions and plasmid incompatibilities in certain instances.

The properties of duplex CTG⅐CAG and CGG⅐CCG, which are involved in the etiology of several hereditary neurodegenerative diseases, were investigated by a variety of methods, including circularization kinetics, apparent helical repeat determination, and polyacrylamide gel electrophoresis. The bending moduli were 1.13 ؋ 10 ؊19 erg⅐cm for CTG and 1.27 ؋ 10 ؊19 erg⅐cm for CGG, ϳ40% less than for random B-DNA. Also, the persistence lengths of the triplet repeat sequences were ϳ60% the value for random B-DNA. However, the torsional moduli and the helical repeats were 2. Eleven human genetic disorders (including fragile X syndrome, myotonic dystrophy, Kennedy's disease, Huntington's disease, spinocerebellar ataxia type 1, dentatorubral-pallidoluysian atrophy, and Friedreich's ataxia) are characterized at the molecular level by the expansion of DNA triplet repeats (CTG, CGG, or AAG) 1 from Ͻ15 copies in normal individuals to scores of copies in affected cases (1-6). In some cases, the CTG and CGG tracts are transcribed into mature mRNAs, whereas the AAG tracts in Friedreich's ataxia are in the first intron of the frataxin gene. The mechanism for expansion is not known, but it may involve slippage of the complementary strands during DNA synthesis (7-10). Expanded alleles undergo further expansions upon passage to offspring and, in some diseases, are associated with the clinical observation called anticipation, whereby the symptoms become more severe in each successive generation and with an earlier age of onset (1-5). This is a novel type of mutation and shows non-mendelian genetic transmission (11,12).Prior investigations suggested that triplet repeat sequences (TRS) 2 do not have the properties of random B-DNA. First, CTG tracts greatly facilitate nucleosome assembly (13-15), which, in turn, may repress transcription. Second, DNA synthesis in vitro pauses at specific loci in fragments containing CTG and CGG (16). Third, long tracts of AAG and AGG form intramolecular triplexes that arrest DNA synthesis (17). Fourth, CTG and CGG migrate up to 30% more rapidly than expected on polyacrylamide gel electrophoresis, whereas their migration is normal on agarose gels (18). Fifth, CTG is preferentially expanded in Escherichia coli compared with the other nine TRS (8). Sixth, the frequency of expansions and deletions in E. coli (7,9,10) is influenced by the direction of replication, suggesting the formation of stable hairpin loops in the lagging strand template or the newly synthesized nascent strand.Conformational investigations were conducted on plasmids and restriction fragments containing CTG and CGG to evaluate their role in the biological behaviors described above. Several methods were applied, including circularization kinetics, apparent helical repeat determinations, the rate of migration through acrylamide and agarose gel electrophoresis, chemical and enzymatic probe analyses, two-dimensional gel electrophoresis, and the induction of an immune response. The analyses indicate that both CTG and CGG exist as fully paired, r...

Several neuromuscular and neurodegenerative diseases are caused by genetically unstable triplet repeat sequences (CTG.CAG, CGG.CCG, or AAG.CTT) in or near the responsible genes. We implemented novel cloning strategies with chemically synthesized oligonucleotides to clone seven of the triplet repeat sequences (GTA.TAC, GAT.ATC, GTT.AAC, CAC.GTG, AGG.CCT, TCG.CGA, and AAG.CTT), and the adjoining paper (Ohshima, K., Kang, S., Larson, J. E., and Wells, R. D.(1996) J. Biol. Chem. 271, 16784-16791) describes studies on TTA.TAA. This approach in conjunction with in vivo expansion studies in Escherichia coli enabled the preparation of at least 81 plasmids containing the repeat sequences with lengths of approximately 16 up to 158 triplets in both orientations with varying extents of polymorphisms. The inserts were characterized by DNA sequencing as well as DNA polymerase pausings, two-dimensional agarose gel electrophoresis, and chemical probe analyses to evaluate the capacity to adopt negative supercoil induced non-B DNA conformations. AAG.CTT and AGG.CCT form intramolecular triplexes, and the other five repeat sequences do not form any previously characterized non-B structures. However, long tracts of TCG.CGA showed strong inhibition of DNA synthesis at specific loci in the repeats as seen in the cases of CTG.CAG and CGG.CCG (Kang, S., Ohshima, K., Shimizu, M., Amirhaeri, S., and Wells, R. D.(1995) J. Biol. Chem. 270, 27014-27021). This work along with other studies (Wells, R. D.(1996) J. Biol. Chem. 271, 2875-2878) on CTG.CAG, CGG.CCG, and TTA.TAA makes available long inserts of all 10 triplet repeat sequences for a variety of physical, molecular biological, genetic, and medical investigations. A model to explain the reduction in mRNA abundance in Friedreich's ataxia based on intermolecular triplex formation is proposed.

Right-handed B and left-handed Z conformations coexist in equilibrium in portions of plasmids in Escherichia coli. The equilibria are influenced by the length of the sequences that undergo the structural transitions and are perturbed by biological processes. The composite results of three types of determinations indicate a supercoil density of -0.025 in vivo. The coexistence of alternative DNA conformations in living cells implies the potential of these structures or their transitions for important functions in genetic regulatory processes.Recent studies (1) Left-handed DNA was demonstrated to exist in E. coli (1) on the basis of the observation that in vitro a target (recognition) site is not methylated by its specific methylase when the site is near (5) or in (6, 7) a left-handed Z helix. However, these enzymes do act on the same target sequences when they exist in a right-handed B structure. The gene for a temperature-sensitive EcoRI methylase (M-EcoRI) was cloned in a pACYC184 derivative to give pRW1602, and conditions were established for its temperature-dependent expression (1). pRW1602 was cotransformed with any one of 10 pBR322 derivative plasmids that contained inserts with different capabilities in vitro of forming Z or cruciform structures, depending on the lengths, orientations, and types of sequences. The excellent correlations between the ability of a sequence to inhibit methylation by M'EcoRI in vivo and in vitro, as well as its relative capacity to adopt a Z-helix in vitro, established the concept of left-handed DNA in vivo.Herein, we have employed a second assay for left-handed DNA in vivo that is based on a change in linking number for plasmids with inserts that undergo conformational transitions. Also, we have substantially extended our previous observations (1) number distribution compared to the culture with chloramphenicol was observed for any of the seven examples studied in this way. The topoisomer distributions were analyzed on 1% agarose gels in TBE buffer (0.09 M Tris/0.09 M boric acid/2.5 mM EDTA, pH 8.3) containing chloroquine (3.5-6.0 ,uM) for 40 hr at 3 V/cm.In Vitro and in Vivo M-EcoRI Studies. In vitro reactions with M-EcoRI as well as in vivo assays with the temperaturesensitive M-EcoRI were performed as described (1). RESULTSLinking Number Assay. When a B-to-Z transition occurs in vivo, the supercoil density -qo in a plasmid increases (i.e., becomes less negative) due to the relaxation process. However, to maintain a constant level of -o-,, DNA gyrase further decreases the linking difference AL to a more negative value AL', until the original -o value is reestablished (9-13). By analyzing the isolated plasmid DNA on agarose gels containing chloroquine (which reverses the B-to-Z transition), those molecules that had contained Z-DNA inside the cell should appear as bands with a more negative linking difference AL' than those that did not contain Z-DNA. In other words, the appearance of a second population of topoisomers (or a complete shift of the whole population) rela...