Nucleic acid triplexes in vivo

Based on in vitro experiments triplex structures have been suggested to play a role in a range of cellular functions, such as transcriptional or translational regulation, interferences with recombination and replication (see Chapter 1.2), post-transcriptional RNA processing and DNA repair. The main focuses of studies investigating triplex structures in vivo were H-DNA and TFOs. Different studies have identified triplex motifs in eukaryotes and prokaryotes by means of computation. Most algorithms search for TFO binding sites (162-164), potential triplex target sites (165), or focus on inverted repeats (166,167) and H-DNA (168,169).

Evidence for the in vivo existence of triplex DNA structures is increasing – immunodetection by triple-helix specific antibodies has been reported (170-172). Those antibodies are able to detect DNA-DNA/DNA and DNA-DNA/RNA (/ indicates Hoogsteen bond) triplex structures (172-175). In addition, different proteins which specifically recognize triplex structures in cells have been identified in human (176), Drosophila (177), yeast (178) and other mammalian cells (179-181). Among those are RecQ helicases (182-184) that actively unwind triplexes in 3’5’ direction, but also heterogeneous ribonucleoproteins (176), intermediate filament proteins (181), high mobility group proteins (182,185,186) and proteins involved in DNA repair (187-189).

Intermolecular triplexes have been used for the artificial regulation of gene expression and may be suitable for therapeutic use (56,190). There are different examples for transcription being influenced by TFO-directed triplex formation in vivo. A mechanism where the triplex formation in the 5’ untranslated region (UTR) shields DNA from duplex targeting proteins such as transcription factors was shown for the ets2 gene in prostate cancer cells (191). In that study, TFOs were designed to overlap the binding site of the transcription factor Sp1, thus triplex formation inhibited transcription. The same principle of transcriptional inhibition was shown for the BCR/ABL locus in human cells (192). In biomedical applications, intermolecular triplexes have been reported to block protein-DNA interactions (193) and

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influence site-directed recombination (194). The possibility of site-specific delivery of target agents via the formation of intermolecular triplexes between the DNA duplex and the TFO has been exploited (195). Using this concept with peptide nucleic acid (PNA) molecules as TFOs different studies showed the introduction of hereditary gene modifications (196,197) and the improvement of the delivery of peptides into the cell nucleus (198). Nucleotide excision repair (NER) factors are able to recognize intermolecular triplex structures (199) and support triplex-induced mutagenesis and recombination events in cells (200,201). Several analysis tools exist for the computational search for TFO binding sites in genomic loci (162,163,168). Putative triplex target sites are over-represented in both prokaryotic and eukaryotic genomes (202,203).

H-DNA is known to induce genetic instability, to have influence on DNA replication and repair and to be involved in transcription (12). Computational studies revealed that natural sequences with the potential to adopt an H-DNA structure are very abundant in mammalian cells (166). Mirror repeats capable of forming H-DNA structures have been found in promoters and coding regions of many genes involved in diseases, such as Friedreich’s ataxia, autosomal dominant polycystic kidney disease (ADPK), fragile X syndrome, spinocerebellar ataxia and muscular dystrophy (204). One well-studied example is Friedreich’s ataxia: Here, H-DNA structures can be induced by expansion of GAA repeats and lead to stalling of the RNA polymerase, thereby silencing the transcription of the frataxin gene (205). The ADPK disease is associated with mutations in the TSC2 and PKD genes.

The proposed mutagenic mechanism involved double strand breaks leading to a replication fork blockade inducing gene conversion by recombination. Interestingly, these genes contain long polypurine/polypyrimidine repeats which are able to form H-DNA and have been shown to be hot-spots for recombination in this region (206). The implication of H-DNA in transcriptional regulation was also studied for the C-MYC oncogene. The H-DNA forming sequence of the C-MYC promoter serves as a cis-acting element downregulating transcription in mammalian cells (207,208). Different studies investigating the role of H-DNA in the regulation of eukaryotic transcription demonstrated either up- (209) or downregulation (210,211) without clarifying specific mechanisms. Genetic instability induced by double-strand breaks adjacent to H-DNA sequences was demonstrated for the C-MYC triplex sequence, but also for model H-DNA sequences in mammalian cells (212). Such double-strand breaks could be induced by replication stalling. In vivo studies revealed that distinct R-type triplex DNA structures can lead to polymerase arrest during elongation of replication, as proposed for the ADPK disease (213,214). Like intermolecular triplexes, intramolecular structures are also able to induce recombination and repair (94). Furthermore, H-DNA sequences have been mapped at recombination hot-spots in mouse myeloma cells (215).

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H-DNA forming sequences inserted into shuttle vectors stimulated recombination events between plasmids in mammalian cells (216). Processes demonstrating recombination between two triplex structures forming at homologous sites have been proposed for H-DNA structures as well (217-219).

Triplex structures in RNA are known to contribute to folding and tertiary structure stability (72), some of them even provide enzymatic or catalytic activity (220,221). Furthermore, they have been reported to cause ribosomal frameshifting during translation. A prominent example is mRNA of the HIV virus which forms an intramolecular triplex inducing -1 ribosomal frameshifting (222,223). Additionally, triplex structures can play a role in chromosomal organization and epigenetics (56). H-DNA formation could provide contact points that interact with non-coding RNAs or cell matrix-associated proteins (224). In addition, the chromatin condensation is influenced by triplex-helices. As triplex structures are less flexible, the nucleosome reconstitution could be affected (225). Schmitz et al. described a TFO-directed triplex which regulates the methylation status of DNA by mediating the recruitment of methyltransferases to promoters (226). DNA methylation plays an important role in epigenetics and is known to influence gene expression and cell differentiation (227,228).

1.2.2.1 Triplexes in prokaryotes

As is the case for G-quadruplexes most of the studies investigating triplexes in vivo were performed in eukaryotes. Information about prokaryotic triplex structures and their functions is rare. Indeed, only few sequences with the potential to form triplex structures were found in prokaryotic species (166). However, long (≥12 nt) oligopurine/oligopyrimidine tracts have been discovered in bacterial genomes near regulatory regions (229), suggesting a functional role. Some studies investigating eukaryotic triplex structures were performed in bacterial cells using plasmids, as they are more convenient model systems. Chemical probing of intracellular DNA showed the formation of H-DNA during transcription of long GC stretches upstream of a promoter in an E. coli plasmid system (230). Triplex formation via addition of TFOs was demonstrated to inhibit transcriptional initiation by the E. coli RNA polymerase in vitro (231,232). However, subsequent in vivo studies were not performed in prokaryotic cells.

The 2.5 kbp long polypyrimidine sequence associated with the ADPK disease, which was found in the human PKD gene (see 1.2.2), has also been investigated in E. coli plasmids. It has been shown to induce double-strand breaks at the H-DNA forming regions which resulted in large scale deletions (233). Furthermore, this sequence activated an SOS

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response and NER in E. coli (234). Two independent studies demonstrated the dimerization of plasmids containing potential triplex forming sequences in E. coli, suggesting a role as interaction point in recombination (235,236). In 1992, Kato et al. showed that triplex DNA inserted in the promoter region of a reporter plasmid expressing β-lactamase resulted in increased lacZ gene expression compared to a control plasmid. They suggested that the triplex structure kept the template in a superhelicity state favorable for gene expression (237). However, when an H-DNA sequence was inserted between the promoter and the coding sequence or directly in the coding region, a downregulation of bacterial gene expression was observed, possibly related to transcriptional regulation (238-240). Although these sequences do not originate from bacteria their influence on bacterial systems implies that triplex structures play a role in bacterial gene regulation, genetic stability and repair mechanisms.

In two subsequent studies, Maher and co-workers investigated so-called PIT (potential intrastrand triplex) elements naturally occurring in E. coli, Synechocystis sp. and H. influenza (10,85). They characterized the PIT motif in E. coli and proposed a triplex structure of the corresponding oligonucleotide. In a follow-up study (85) they wanted to elucidate the function of PIT elements. Although they showed that, depending on the processability of the polymerase, PIT elements are able to block DNA polymerase elongation in vitro, they found no effect in in vivo studies. The PIT elements showed no promoter and terminator activity, had no effect on RNA polymerase and reverse transcriptase and did not interfere with conjugation.

In a different study, a bacterial protein interacting with triplex DNA was described. The protein TnsC, regulating the transposition of transposon Tn7 was observed to detect triplex DNA. Triplex recognition then leads to specific insertion of the transposon adjacent to intra- or intermolecular pyrimidine triplex motifs (241).