A bulge binding agent with novel wedge-shape topology for stimulation of DNA triplet repeat strand slippage synthesis

Expansion of DNA repeat sequences is associated with many human genetic diseases. Bulged DNA structures have been implicated as intermediates in DNA slippage within the DNA repeat regions. Herein a bulge binding agent with novel wedge-shape topology of the aromatic moiety was designed and synthesized. The compound-bulge DNA interactions were characterized via UV melting experiments, circular dichroism and were quantitated by surface plasmon resonance with K(d) of 41.5 microM. This compound showed remarkable stimulation for DNA triplet repeat strand slippage synthesis in vitro.     

Stimulation on DNA triplet repeat strand slippage synthesis by the designed spirocycles

The designed simpler chiral spirocyclic helical compounds that mimic the molecular architecture of the DNA bulge binder NCSi-gb have been prepared. It has been found that the synthesized spirocyclic compounds have strong stimulation effect on DNA slippage synthesis. Their stimulation activities on DNA strand slippage suggest that they may bind to or induce the formation of a non Watson-Crick structure during in vitro replication of DNA triplet repeats.     

Stimulation of DNA strand slippage synthesis by a bulge binding synthetic agent

It has been postulated that bulged structures may be intermediates in the DNA strand slippage synthesis associated with the expansion of nucleotide repeats in various neurodegenerative diseases and cancer. To probe the possible role of bulged structures in this process, we have synthesized a wedge-shaped spirocyclic molecule, DDI (double-decker intercalator), on the basis of our earlier work with the bulge-specific derivative prepared from the enediyne antitumor antibiotic neocarzinostatin chromophore. Using a series of primers/templates containing nucleotide repeats [(AAT)(3)/(ATT)(5), (ATT)(3)/(AAT)(5), (CAG)(3)/(CTG)(5), (CA)(4)C/(GT)(7)G, (GT)(4)G/(CA)(7)C, T(9)/A(30), T(20)/A(30)] with the Klenow fragment of Escherichia coli DNA polymerase I, we find that DDI markedly enhances the formation of long DNA products, whose synthesis would require strand slippage to occur. DDI-induced slippage synthesis is more pronounced as the incubation proceeds and at limiting enzyme levels. The gel band pattern of the synthesized DNA products reflects the particular nucleotide repeat unit and is not altered by DDI. The lack of any drug effect on primer extension on M13 DNA and heteropolymeric 62-mer templates, where strand slippage is much less likely to occur, suggests that stimulation of slippage synthesis by DDI is not due to a direct effect on the enzyme. By contrast, other DNA-binding agents, such as ethidium bromide, distamycin, and doxorubicin, inhibit the formation of slippage-induced DNA products, but this block can be overcome by DDI, presumably by its destabilizing duplex DNA-binding sites for these other agents. We propose that DDI binds to or induces the formation of a bulge or related structure, which promotes DNA strand slippage and its consequent expansion of nucleotide repeats during replication by DNA polymerase I and that this action provides insight into the development of agents that interfere with nucleotide expansions found in various disease states.     

Impact of bulge loop size on DNA triplet repeat domains: Implications for DNA repair and expansion

Repetitive DNA sequences exhibit complex structural and energy landscapes, populated by metastable, noncanonical states, that favor expansion and deletion events correlated with disease phenotypes. To probe the origins of such genotype–phenotype linkages, we report the impact of sequence and repeat number on properties of (CNG) repeat bulge loops. We find the stability of duplexes with a repeat bulge loop is controlled by two opposing effects; a loop junction‐dependent destabilization of the underlying double helix, and a self‐structure dependent stabilization of the repeat bulge loop. For small bulge loops, destabilization of the underlying double helix overwhelms any favorable contribution from loop self‐structure. As bulge loop size increases, the stabilizing loop structure contribution dominates. The role of sequence on repeat loop stability can be understood in terms of its impact on the opposing influences of junction formation and loop structure. The nature of the bulge loop affects the thermodynamics of these two contributions differently, resulting in unique differences in repeat size‐dependent minima in the overall enthalpy, entropy, and free energy changes. Our results define factors that control repeat bulge loop formation; knowledge required to understand how this helix imperfection is linked to DNA expansion, deletion, and disease phenotypes. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 1–12, 2014.     

Inhibition of DNA synthesis facilitates expansion of low‐complexity repeats

Simple DNA repeats (trinucleotide repeats, micro‐ and minisatellites) are prone to expansion/contraction via formation of secondary structures during DNA synthesis. Such structures both inhibit replication forks and create opportunities for template‐primer slippage, making these repeats unstable. Certain aspects of simple repeat instability, however, suggest additional mechanisms of replication inhibition dependent on the primary DNA sequence, rather than on secondary structure formation. I argue that expanded simple repeats, due to their lower DNA complexity, should transiently inhibit DNA synthesis by locally depleting specific DNA precursors. Such transient inhibition would promote formation of secondary structures and would stabilize these structures, facilitating strand slippage. Thus, replication problems at simple repeats could be explained by potentiated toxicity, where the secondary structure‐driven repeat instability is enhanced by DNA polymerase stalling at the low complexity template DNA. This minireview is dedicated to the FASEB‐2012 meeting “Dynamic DNA Structures in Biology”, organized by Nancy Maizels and Sergei Mirkin.     

Hairpin formation in Friedreich's ataxia triplet repeat expansion

Triplet repeat tracts occur throughout the human genome. Expansions of a (GAA)(n)/(TTC)(n) repeat tract during its transmission from parent to child are tightly associated with the occurrence of Friedreich's ataxia. Evidence supports DNA slippage during DNA replication as the cause of the expansions. DNA slippage results in single-stranded expansion intermediates. Evidence has accumulated that predicts that hairpin structures protect from DNA repair the expansion intermediates of all of the disease-associated repeats except for those of Friedreich's ataxia. How the latter repeat expansions avoid repair remains a mystery because (GAA)(n) and (TTC)(n) repeats are reported not to self-anneal. To characterize the Friedreich's ataxia intermediates, we generated massive expansions of (GAA)(n) and (TTC)(n) during DNA replication in vitro using human polymerase beta and the Klenow fragment of Escherichia coli polymerase I. Electron microscopy, endonuclease cleavage, and DNA sequencing of the expansion products demonstrate, for the first time, the occurrence of large and growing (GAA)(n) and (TTC)(n) hairpins during DNA synthesis. The results provide unifying evidence that predicts that hairpin formation during DNA synthesis mediates all of the disease-associated, triplet repeat expansions.     

DNA base excision repair: a mechanism of trinucleotide repeat expansion

The expansion of trinucleotide repeat (TNR) sequences in human DNA is considered to be a key factor in the pathogenesis of more than 40 neurodegenerative diseases. TNR expansion occurs during DNA replication and also, as suggested by recent studies, during the repair of DNA lesions produced by oxidative stress. In particular, the oxidized guanine base 8-oxoguanine within sequences containing CAG repeats may induce formation of pro-expansion intermediates through strand slippage during DNA base excision repair (BER). In this article, we describe how oxidized DNA lesions are repaired by BER and discuss the importance of the coordinated activities of the key repair enzymes, such as DNA polymerase β, flap endonuclease 1 (FEN1) and DNA ligase, in preventing strand slippage and TNR expansion.     

Replication slippage involves DNA polymerase pausing and dissociation

Genome rearrangements can take place by a process known as replication slippage or copy-choice recombination. The slippage occurs between repeated sequences in both prokaryotes and eukaryotes, and is invoked to explain microsatellite instability, which is related to several human diseases. We analysed the molecular mechanism of slippage between short direct repeats, using in vitro replication of a single-stranded DNA template that mimics the lagging strand synthesis. We show that slippage involves DNA polymerase pausing, which must take place within the direct repeat, and that the pausing polymerase dissociates from the DNA. We also present evidence that, upon polymerase dissociation, only the terminal portion of the newly synthesized strand separates from the template and anneals to another direct repeat. Resumption of DNA replication then completes the slippage process.     

Generation of long tracts of disease-associated DNA repeats

The generation of long uninterrupted DNA repeats is important for the study of repeat instability associated with several human genetic diseases, including myotonic dystrophy type 1. However, obtaining defined lengths of long repeats in vitro has been problematic. Strand slippage and/or DNA secondary structure formation may prevent efficient ligation. For example, a purified (CTG)140.(CAG)140 repeat fragment containing 4-bp AGCA/TGCT overhanging ends ligated poorly using T4 or Escherichia coli DNA ligase, although limited repeat ligation occurred using thermostable DNA ligase. Here we describe a general procedure for ligating multimers of DNA repeats. Multimers are efficiently ligated when slippage is prevented or when DNA repeats contain a single G/C overhang. A cloning vector is designed from which pure repeat fragments containing a G/C overhang can be generated for further ligation. (CAG)n.(CTG)n DNA molecules longer than 800 bp were generated using this approach. This approach also worked for (GAA)n.(TTC)n, (CCTG)n-(CAGG)n, and (ATTCT)n.(AGAAT)n tracts associated with Friedreich ataxia, DM2, and spinocerebellar ataxia type 10, respectively.     

Solution structure of a wedge-shaped synthetic molecule at a two-base bulge site in DNA

The solution structure of the complex formed between an oligonucleotide containing a two-base bulge (5'-CACGCAGTTCGGAC.5'-GTCCGATGCGTG) and DDI, a designed synthetic agent, has been elucidated using high-resolution NMR spectroscopy and restrained molecular dynamic simulation. DDI, which has been found to modulate DNA strand slippage synthesis by DNA polymerase I [Kappen, L. S., Xi, Z., Jones, G. B., and Goldberg, I. H. (2003) Biochemistry 42, 2166-2173], is a wedge-shaped spirocyclic molecule whose aglycone structure closely resembles that of the natural product, NCSi-gb, which strongly binds to an oligonucleotide containing a two-base bulge. Changes in chemical shifts of the DNA upon complex formation and intermolecular NOEs between DDI and the bulged DNA duplex indicate that agent specifically binds to the bulge site of DNA. The benzindanone moiety of DDI intercalates via the minor groove into the G7-T8-T9.A20 pocket, which consists of a helical base pair and two unpaired bulge bases, stacking with the G7 and A20 bases. On the other hand, the dihydronaphthalenone and aminoglycoside moieties are positioned in the minor groove. The aminoglycoside, which is attached to spirocyclic ring, aligns along the A20T21G22 sequence of the nonbulged strand, while the dihydronaphthalenone, which is restrained by the spirocyclic structure, is positioned near the G7-T8-T9 bulge site. The aminoglycoside is closely aligned with the dihydronaphthalenone, preventing its intercalation into the bulge site. In the complex, the unpaired purine (G7) is intrahelical and stacks with the intercalating moiety of DDI, whereas the unpaired pyrimidine (T8) is extrahelical. The structure of the complex formed by binding of the synthetic agent to the two-base bulged DNA reveals a binding mode that differs in important details from that of the natural product, explaining the different binding specificity for the bulge sites of DNA. The structure of the DDI-bulged DNA complex provides insight into the structure-binding affinity relationship, providing a rational basis for the design of specific, high-affinity probes of the role of bulged nucleic acid structures in various biological processes.     

Progressive myoclonus epilepsy [EPM1] repeat d(CCCCGCCCCGCG)n forms folded hairpin structures at physiological pH.

The secondary structure of DNA has been shown to be an important component in the mechanism of expansion of the trinucleotide repeats that are associated with many neurodegenerative disorders. Recently, expansion of a dodecamer repeat, (CCCCGCCCCGCG)n upstream of cystatin B gene has been shown to be the most common mutation associated with Progressive Myoclonus Epilepsy (EPM1) of Unverricht-Lundborg type. We have investigated structure of oligonucleotides containing one, two and three copies of the EPM1 repeat sequences at physiological pH. CD spectra and anomalous faster gel electrophoretic mobilty indicates formation of intramolecularly folded structures that are formed independent of concentration. Hydroxylamine probing allowed us to identify the C residues that are involved in C.G base pairing. P1 nuclease studies elucidated the presence of unpaired regions in the folded back structures. UV melting studies show biphasic melting curves for the oligonucleotides containing two and three EPM1 repeats. Our data suggests multiple hairpin structures for two and three repeat containing oligonucleotides. In this paper we show that oligonucleotides containing EPM1 repeat adopt secondary structures that may facilitate strand slippage thereby causing the expansion.     

Structural analysis of slipped-strand DNA (S-DNA) formed in (CTG)n. (CAG)n repeats from the myotonic dystrophy locus.

The mechanism of disease-associated trinucleotide repeat length variation may involve slippage of the triplet-containing strand at the replication fork, generating a slipped-strand DNA structure. We recently reported formation in vitro of slipped-strand DNA (S-DNA) structures when DNAs containing triplet repeat blocks of myotonic dystrophy or fragile X diseases were melted and allowed to reanneal to form duplexes. Here additional evidence is presented that is consistent with the existence of S-DNA structures. We demonstrate that S-DNA structures can form between two complementary strands containing equal numbers of repeats. In addition, we show that both the propensity for S-DNA formation and the structural complexity of S-DNAs formed increase with increasing repeat length. S-DNA structures were also analyzed by electron microscopy, confirming that the two strands are slipped out of register with respect to each other and confirming the structural polymorphism expected within long tracts of trinucleotide repeats. For (CTG)50.(CAG)50 two distinct populations of slipped structures have been identified: those involving </=10 repeats per slippage, which appear as bent/kinked DNA molecules, and those involving >10 repeats, which have multiple loops or hairpins indicative of complex alternative DNA secondary structures.     

Structural features of trinucleotide repeats associated with DNA expansion.

The mechanism of DNA expansion is not well understood. Recent evidence from genetic, in vivo, and in vitro studies has suggested a link between the formation of alternative DNA secondary structures by trinucleotide repeat tracts and their propensity to undergo expansion. This review will focus on structural features and the mechanism of expansion relevant to human disease.     

Triplet repeat expansion generated by DNA slippage is suppressed by human flap endonuclease 1

Human flap endonuclease 1 (h-FEN1) mutations have dramatic effects on repeat instability. Current models for repeat expansion predict that h-FEN1 protein prevents mutations by removing 5'-flaps generated at ends of Okazaki fragments by strand displacement synthesis. The models propose that hairpin formations within flaps containing repeats enable them to escape h-FEN1 cleavage. Friedreich's ataxia is caused by expansion mutations in a d(GAA)n repeat tract. Single-stranded d(GAA)n repeat tracts, however, do not form stable hairpins until the repeat tracts are quite long. Therefore, to understand how d(GAA)n repeat expansions survive h-FEN1 activity, we determined the effects of h-FEN1 on d(GAA)n repeat expansion during replication of a d(TTC)n repeat template. Replication initiated within the repeat tract generated significant expansion that was suppressed by the addition of h-FEN1 at the start of replication. The ability of h-FEN1 to suppress expansion implies that DNA slippage generates a 5'-flap in the nascent strand independent of strand displacement synthesis by an upstream polymerase. Delaying the addition of h-FEN1 to the replication reaction abolished the ability of h-FEN1 ability to suppress d(GAA)n repeat expansion products of all sizes, including sizes unable to hairpin. Use of model substrates demonstrated that h-FEN1 cleaves d(GAA)n 5'-flaps joined to double-stranded nonrepeat sequences but not those joined to double-stranded repeat tracts. The results provide evidence that, given the opportunity, short d(GAA)n repeat expansion products rearrange from 5'-flaps to stable internal loops inside the repeat tract. Long expansion products are predicted to form hairpinned flaps and internal loops. Once formed, these DNA conformations resist h-FEN1. The biological implications of the results are discussed.     

DNA ligase I competes with FEN1 to expand repetitive DNA sequences in vitro

Repeat sequences in various genomes undergo expansion by poorly understood mechanisms. By using an oligonucleotide system containing such repeats, we recapitulated the last steps in Okazaki fragment processing, which have been implicated in sequence expansion. A template containing either triplet or tandem repeats was annealed to a downstream primer containing complementary repeats at its 5'-end. Overlapping upstream primers, designed to strand-displace varying numbers of repeats in the downstream primer, were annealed. Human DNA ligase I joined overlapping segments of repeats generating an expansion product from the primer strands. Joining efficiency decreased with repeat length. Flap endonuclease 1 (FEN1) cleaved the displaced downstream strand and together with DNA ligase I produced non-expanded products. However, both expanded and non-expanded products formed irrespective of relative nuclease and ligase concentrations tested or enzyme addition order, suggesting the pre-existence and persistence of intermediates leading to both outcomes. FEN1 activity decreased with the length of repeat segment displaced presumably because the flap forms structures that inhibit cleavage. Increased MgCl(2) disfavored ligation of substrate intermediates that result in expansion products. Examination of expansion in vitro enables dissection of substrate and replication enzyme dynamics on repeat sequences.     

Force-induced DNA slippage

DNA containing repetitive sequences displays richer dynamics than heterogeneous sequences. In the genome the number of repeat units of repetitive sequences, known as microsatellites, may vary during replication by DNA slippage and their expansion gives rise to serious disorders. We studied the mechanical properties of repetitive DNA using dynamic force spectroscopy and found striking differences compared with ordinary heterogeneous sequences. Repetitive sequences dissociate at lower forces and elongate above a certain threshold force. This yield force was found to be rate dependent. Following the rapid stretching of the DNA duplex, the applied force relaxes by stepwise elongation of this duplex. Conversely, contraction of the DNA duplex can be observed at low forces. The stepwise elongation and shortening is initiated by single slippage events, and single-molecule experiments might help to explain the molecular mechanisms of microsatellites formation. In addition to the biological importance, the remarkable properties of repetitive DNA can be useful for different nanomechanical applications.     

Mechanism of in vitro expansion of long DNA repeats: effect of temperature, repeat length, repeat sequence, and DNA polymerases.

Studies of sequence repeat expansions from duplexes consisting of DNA repeat sequences greater than three bases are currently lacking. These studies are needed in order to gain a better understanding of DNA expansions in general and as a first step in understanding expansions of longer sequence repeats that have been implicated in human diseases. We have undertaken an in vitro study of tetranucleotide, hexanucleotide, and octanucleotide repeat expansions from short DNA duplexes using Taq DNA polymerase. Expansions of hexanucleotide repeats were also studied with the Klenow fragment of DNA polymerase I and with T4 DNA polymerase. Studies with Taq DNA polymerase show that expansions occur more readily as the length of the repeat sequence decreases but are generally more efficient at reaction temperatures closer to the melting point of the starting duplex. A mechanism for the observed expansions with Taq DNA polymerase is proposed that does not invoke strand slippage or DNA structure. Studies at 37 degrees C with Klenow pol I and T4 DNA polymerase indicate that the template-switching and/or strand-displacement activities of the polymerases used can play a major role in the apparent in vitro expansions of short repetitive DNA duplexes.     

Energetic coupling between clustered lesions modulated by intervening triplet repeat bulge loops: Allosteric implications for DNA repair and triplet repeat expansion

Clusters of closely spaced oxidative DNA lesions present challenges to the cellular repair machinery. When located in opposing strands, base excision repair (BER) of such lesions can lead to double strand DNA breaks (DSB). Activation of BER and DSB repair pathways has been implicated in inducing enhanced expansion of triplet repeat sequences. We show here that energy coupling between distal lesions (8oxodG and/or abasic sites) in opposing DNA strands can be modulated by a triplet repeat bulge loop located between the lesion sites. We find this modulation to be dependent on the identity of the lesions (8oxodG vs. abasic site) and the positions of the lesions (upstream vs. downstream) relative to the intervening bulge loop domain. We discuss how such bulge loop‐mediated lesion crosstalk might influence repair processes, while favoring DNA expansion, the genotype of triplet repeat diseases. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 355–369, 2010. This article was originally published online as an acceptedpreprint. The “Published Online” date corresponds to the preprint version. You can reqest a copy of the preprint byemailing the Biopolymers editorial office at biopolymers@wiley.com     

Interlocked mismatch-aligned arrowhead DNA motifs.

BACKGROUND: Triplet repeat sequences are of considerable biological importance as the expansion of such tandem arrays can lead to the onset of a range of human diseases. Such sequences can self-pair via mismatch alignments to form higher order structures that have the potential to cause replication blocks, followed by strand slippage and sequence expansion. The all-purine d(GGA)n triplet repeat sequence is of particular interest because purines can align via G.G, A.A and G.A mismatch formation. RESULTS: We have solved the structure of the uniformly 13C,15N-labeled d(G1-G2-A3-G4-G5-A6-T7) sequence in 10 mM Na+ solution. This sequence adopts a novel twofold-symmetric duplex fold where interlocked V-shaped arrowhead motifs are aligned solely via interstrand G1.G4, G2.G5 and A3.A6 mismatch formation. The tip of the arrowhead motif is centered about the p-A3-p step, and symmetry-related local parallel-stranded duplex domains are formed by the G1-G2-A3 and G4-G5-A6 segments of partner strands. CONCLUSIONS: The purine-rich (GGA)n triplet repeat sequence is dispersed throughout the eukaryotic genome. Several features of the arrowhead duplex motif for the (GGA)2 triplet repeat provide a unique scaffold for molecular recognition. These include the large localized bend in the sugar-phosphate backbones, the segmental parallel-stranded alignment of strands and the exposure of the Watson-Crick edges of several mismatched bases.