Intramolecular folding of a fragment of the cytosine-rich strand of telomeric DNA into an i-motif.

In the recently discovered i-motif, four stretches of cytosine form two parallel-stranded duplexes whose C.C+ base pairs are fully intercalated. The i-motif may be recognized by characteristic Overhauser cross-peaks of the proton NMR spectrum, reflecting short H1'-H1' distances across the minor groove, and short internucleotide amino-proton-H2'/H2" across the major groove. We report the observation of such cross-peaks in the spectra of a fragment of the C-rich telomeric strand of vertebrates, d[CCCTAA]3CCC. The spectra also demonstrate that the cytosines are base-paired and that proton exchange is very slow, as reported previously for the i-motif. From UV absorbance and gel chromatography measurements, we assign these properties to an i-motif which includes all or nearly all the cytosines, and which is formed by intramolecular folding at slightly acid or neutral pH. A fragment of telomeric DNA of Tetrahymena, d[CCCCAA]3CCCC, has the same properties. Hence four consecutive C stretches of a C-rich telomeric strand can fold into an i-motif. Hypothetically, this could occur in vivo.     

Studying the Influence of the Pyrene Intercalator TINA on the Stability of DNA i-Motifs

Certain cytosine-rich (C-rich) DNA sequences can fold into secondary structures as four-stranded i-motifs with hemiprotonated base pairs. Here we synthesized C-rich TINA-intercalating oligonucleotides by inserting a nonnucleotide pyrene moiety between two C-rich regions. The stability of their i-motif structures was studied by using UV melting temperature measurements and circular dichroism spectra at different pH values under noncrowding and crowding conditions (20% poly(ethylene glycol)). When TINA ((R)-3-((4-(1-pyrenylethynyl)benzyl)oxy) propane-1,2-diol) is inserted, the oligonucleotides could form an i-motif at a higher pH than observed for the corresponding wildtype oligonucleotide.     

Tetraplex formation of surface-immobilized human telomere sequence probed by surface plasmon resonance using single-stranded DNA binding protein

Many sequences in genomic DNA are able to form unique tetraplex structures. Such structures are involved in a variety of important cellular processes and are emerging as a new class of therapeutic targets for cancers and other diseases. Screening for molecules targeting the tetraplex structure has been explored using such sequences immobilized on solid surfaces. Immobilized nucleic acids, in certain situations, may better resemble the molecules under in vivo conditions. In this report, we studied the formation of tetraplex structure of both the G-rich and C-rich strands of surface-immobilized human telomere sequence by surface plasmon resonance using the single-stranded DNA binding protein from Escherichia coli as probe. We demonstrate how the formation of G-quadruplex and i-motif could be probed under various conditions by this sequence-universal method. Our results also show that immobilization destabilized the tetraplex structure.     

Intramolecular i-motif structure at acidic pH for progressive myoclonus epilepsy (EPM1) repeat d(CCCCGCCCCGCG)n.

The most common mutation associated with Progressive Myoclonus Epilepsy (EPM1) of Unverricht-Lundberg type is the expansion of a dodecamer repeat, d(CCCCGCCCCGCG)n. We show that the C-rich strand of this repeat (2-3 copies) forms intercalated i-motif structure at acidic pH as judged by CD spectroscopy and anomalous gel electrophoretic mobility. The stability of the structure increases with the increase in the length of the repeat. Transient formation of stable, folded back structure like i-motif could play an important role in the mechanism of expansion of this repeat.     

Heterogeneous nuclear ribonucleoprotein A3 binds single-stranded telomeric DNA and inhibits telomerase extension in vitro

Telomeres are dynamic DNA-protein complexes at the end of linear chromosomes. Maintenance of functional telomeres is required for chromosome stability, and to avoid the activation of DNA damage response pathway and cell cycle arrest. Telomere-binding proteins play crucial roles in the maintenance of functional telomeres. In this study, we employed affinity pull-down and proteomic approach to search for novel proteins that interact with the single-stranded telomeric DNA. The proteins identified by two-dimensional gel electrophoresis were further characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and MALDI-TOF-TOF tandem MS. Among the five identified proteins, we report here the biochemical properties of a novel protein, hnRNP A3. The purified hnRNP A3 bound specifically to G-rich strand, but not to C-rich strand or double-stranded telomeric DNA. The RRM1 (RNA recognition motif 1) domain, but not RRM2, of hnRNP A3 is sufficient to confer specific binding to the telomeric sequence. In addition, we present evidence that hnRNP A3 can inhibit telomerase extension in vitro. These biochemical properties of hnRNP A3 suggest that hnRNP A3 can participate in telomere regulation in vivo.     

The RNA i-motif

Oligodeoxynucleotides with stretches of cytidine residues associate into a four-stranded structure, the i-motif, in which two head-to-tail, intercalated, parallel-stranded duplexes are held together by hemiprotonated C.C+ pairs. We have investigated the possibility of forming an i-motif structure with C-rich ribonucleic acids. The four C-rich RNAs studied, r(UC5), r(C5), r(C5U) and r(UC3), associate into multiple intercalated structures at acidic pH. r(UC5) forms two i-motif structures that differ by their intercalation topologies. We report on a structural study of the main form and we analyze the small conformational differences found by comparison with the DNA i-motif. The stacking topology of the main structure avoids one of the six 2'-OH/2'-OH repulsive contacts expected in a fully intercalated structure. The C3'-endo pucker of the RNA sugars and the orientation of the intercalated C.C+ pairs result in a modest widening of the narrow grooves at the steps where the hydroxyl groups are in close contact. The free energy of the RNA i-motif, on average -4 kJ mol(-1) per C.C+ pair, is half of the value found in DNA i-motif structures.     

Rice proteins that bind single-stranded G-rich telomere DNA

In this work, we have identified and characterized proteins in rice nuclear extracts that specifically bind the single-stranded G-rich telomere sequence. Three types of specific DNA-protein complexes (I, II, and III) were identified by gel retardation assays using synthetic telomere substrates consisting of two or more single-stranded TTTAGGG repeats and rice nuclear extracts. Since each complex has a unique biochemical property and differs in electrophoretic mobility, at least three different proteins interact with the G-rich telomere sequences. These proteins are called rice G-rich telomere binding protein (RGBP) and none of them show binding affinity to double-stranded telomere repeats or single-stranded C-rich sequence. Changing one or two G's to C's in the TTTAGGG repeats abolishes binding activity. RGBPs have a greatly reduced affinity for human and Tetrahymena telomeric sequence and do not efficiently bind the cognate G-rich telomere RNA sequence UUUAGGG. Like other telomere binding proteins, RGBPs are resistant to high salt concentrations. RNase sensitivity of the DNA-protein interactions was tested to investigate whether an RNA component mediates the telomeric DNA-protein interaction. In this assay, we observed a novel complex (complex III) in gel retardation assays which did not alter the mobilities or the band intensities of the two pre-existing complexes (I and II). The complex III, in addition to binding to telomeric sequences, has a binding affinity to rice nuclear RNA, whereas two other complexes have a binding affinity to only single-stranded G-rich telomere DNA. Taken together, these studies suggest that RGBPs are new types of telomere-binding proteins that bind in vitro to single-stranded G-rich telomere DNA in the angiosperms.     

ST-1, a 39-kilodalton protein in Trypanosoma brucei, exhibits a dual affinity for the duplex form of the 29-base-pair subtelomeric repeat and its C-rich strand.

In our attempt to identify telomere region-binding proteins in Trypanosoma brucei, we identified ST-1, a polypeptide with novel features. ST-1 was chromatographically purified from S-100 cell extracts and was renatured from a sodium dodecyl sulfate-protein gel as a 39-kDa polypeptide. It forms a specific complex with the trypanosome telomere repeats of TTAGGG, but more significantly, it shows a higher affinity for the 29-bp subtelomere repeats of T. brucei. These 29-mer boxes are a large tandem series of telomere-derived repeats which separate the simple telomere DNA from middle-repetitive telomere-associated sequences on many chromosomes. ST-1 is the first example of a protein binding within such large repetitive subtelomere elements in trypanosomes or other organisms. ST-1 is also novel in that it has a selective affinity for the C-rich strands of both the subtelomeric 29-mer and the telomere repeats, comparable to that for the duplex form of the respective repeats. All previously described telomere-binding proteins have affinity for only the duplex form or for the G-rich strand. This C-rich strand binding specificity of ST-1 may provide insight into this protein's mechanism of binding in vivo.     

Kinetic hybrid i-motifs: intercepting DNA with RNA to form a DNA(2)-RNA(2) i-motif

We have constructed and characterized a long-lived hybrid DNA(2)-RNA(2) i-motif that is kinetically formed by mixing equivalent amount of C-rich RNA (R) and C-rich DNA (D). Circular dichroism shows that these hybrids are distinct from their parent DNA(4) or RNA(4) i-motif. pH dependent CD and UV thermal melting experiments showed that the complexes were maximally stable at pH 4.5, the pK(a) of cytosine, consistent with the complex being held by CH(+)-C base pairs. Fluorescence studies confirmed their tetrameric nature and established the relative strand polarities of the RNA and DNA strands in the complex. These showed that in a hybrid D(2)R(2) i-motif two DNA strands occupy one narrow groove and the two RNA strands occupy the other. This suggests that even the sugar-sugar interactions are highly specific. Interestingly, this hybrid slowly disproportionates into DNA(4) i-motifs and ssRNA which would be valuable to study intermediates in DNA(4) i-motif formation.     

Hybrid DNA i-motif: Aminoethylprolyl-PNA (pC5) enhance the stability of DNA (dC5) i-motif structure

This report describes the synthesis of C-rich sequence, cytosine pentamer, of aep-PNA and its biophysical studies for the formation of hybrid DNA:aep-PNAi-motif structure with DNA cytosine pentamer (dC₅) under acidic pH conditions. Herein, the CD/UV/NMR/ESI-Mass studies strongly support the formation of stable hybrid DNA i-motif structure with aep-PNA even near acidic conditions. Hence aep-PNA C-rich sequence cytosine could be considered as potential DNA i-motif stabilizing agents in vivo conditions.     

Identification and characterization of inosine monophosphate dehydrogenase from Halobacterium salinarum

Extremely halophilic Archaea, Halobacterium salinarum live in hypersaline habitats and maintain an osmotic balance of their cytoplasm by accumulating high concentrations of salt (mainly KCl). Therefore, their enzymes adapted to high NaCl concentrations offer a multitude of acutal or potential applications such as biocatalysts in the presence of high salt concentrations. In this study, the protein expression profile of H. salinarum cultured under different NaCl concentrations (3.5 M, 4.3 M, and 6.0 M) was investigated using two-dimensional gel electrophoresis (2-DE). As a result of 2-DE, the protein spots concentrated in acidic range at pH 3-10 were separated effectively using pH 3.5-4.5 ultrazoom IPG DryStrips. The proteins which proved to be upregulated or downregulated in 2-DE gel were digested with trypsin and identified with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and electrospray ionization quadrupole (ESI-Q) TOF-mass spectrometry. Most proteins were identified as known annotated proteins based on sequence homology and few as unknown hypothetical proteins. Among proteins identified, an enzyme named inosine monophosphate dehydrogenase (IMPDH) was selected based on the possibility of its industrial application. IMPDH gene (1.6 kb fragment) expected to exist in H. salinarum was amplified by polymerase chain reaction (PCR) and expressed in Escherichia coli strain, BL21 (DE3) using a pGEX-KG vector. Recombinant IMPDH purified from H. salinarum has a higher activity in the presence of salt than in the absence of salt.     

C. elegans telomeres contain G-strand and C-strand overhangs that are bound by distinct proteins

Single-strand extensions of the G strand of telomeres are known to be critical for chromosome-end protection and length regulation. Here, we report that in C. elegans, chromosome termini possess 3' G-strand overhangs as well as 5' C-strand overhangs. C tails are as abundant as G tails and are generated by a well-regulated process. These two classes of overhangs are bound by two single-stranded DNA binding proteins, CeOB1 and CeOB2, which exhibit specificity for G-rich or C-rich telomeric DNA. Strains of worms deleted for CeOB1 have elongated telomeres as well as extended G tails, whereas CeOB2 deficiency leads to telomere-length heterogeneity. Both CeOB1 and CeOB2 contain OB (oligo-saccharide/oligo-nucleotide binding) folds, which exhibit structural similarity to the second and first OB folds of the mammalian telomere binding protein hPOT1, respectively. Our results suggest that C. elegans telomere homeostasis relies on a novel mechanism that involves 5' and 3' single-stranded termini.     

Human telomeric DNA: G-quadruplex,i-motif and Watson-Crick double helix

Human telomeric DNA composed of (TTAGGG/CCCTAA)n repeats may form a classical Watson-Crick double helix. Each individual strand is also prone to quadruplex formation: the G-rich strand may adopt a G-quadruplex conformation involving G-quartets whereas the C-rich strand may fold into an i-motif based on intercalated C*C+ base pairs. Using an equimolar mixture of the telomeric oligonucleotides d[AGGG(TTAGGG)3] and d[(CCCTAA)3CCCT], we defined which structures existed and which would be the predominant species under a variety of experimental conditions. Under near-physiological conditions of pH, temperature and salt concentration, telomeric DNA was predominantly in a double-helix form. However, at lower pH values or higher temperatures, the G-quadruplex and/or the i-motif efficiently competed with the duplex. We also present kinetic and thermodynamic data for duplex association and for G-quadruplex/i-motif unfolding.     

Sequence-specific binding of Taz1p dimers to fission yeast telomeric DNA

The fission yeast (Schizosaccharomyces pombe) taz1 gene encodes a telomere-associated protein. It contains a single copy of a Myb-like motif termed the telobox that is also found in the human telomere binding proteins TRF1 and TRF2, and Tbf1p, a protein that binds to sequences found within the sub-telomeric regions of budding yeast (Saccharomyces cerevisiae) chromosomes. Taz1p was synthesised in vitro and shown to bind to a fission yeast telomeric DNA fragment in a sequence specific manner that required the telobox motif. Like the mammalian TRF proteins, Taz1p bound to DNA as a preformed homodimer. The isolated Myb-like domain was also capable of sequence specific DNA binding, although with less specificity than the full-length dimer. Surprisingly, a protein extract produced from a taz1–fission yeast strain still contained the major telomere binding activity (complex I) we have characterised previously, suggesting that there could be other abundant telomere binding proteins in fission yeast. One candidate, SpX, was also synthesised in vitro, but despite the presence of two telobox domains, no sequence specific binding to telomeric DNA was detected.     

Cationic porphyrins promote the formation of i-motif DNA and bind peripherally by a nonintercalative mechanism

Telomeric C-rich strands can form a noncanonical intercalated DNA structure known as an i-motif. We have studied the interactions of the cationic porphyrin 5,10,15,20-tetra-(N-methyl-4-pyridyl)porphine (TMPyP4) with the i-motif forms of several oligonucleotides containing telomeric sequences. TMPyP4 was found to promote the formation of the i-motif DNA structure. On the basis of (1)H NMR studies, we have created a model of the i-motif-TMPyP4 complex that is consistent with all the available experimental data. Two-dimensional NOESY data prompted us to conclude that TMPyP4 binds specifically to the edge of the intercalated DNA core by a nonintercalative mechanism. Since we have shown that TMPyP4 binds to and stabilizes the G-quadruplex form of the complementary G-rich telomeric strand, this study raises the intriguing possibility that TMPyP4 can trigger the formation of unusual DNA structures in both strands of the telomeres, which may in turn explain the recently documented biological effects of TMPyP4 in cancer cells.     

Characterization of nucleoprotein complexes in plants with human-type telomere motifs.

A conserved feature of telomeres is the 3'-overhang of their G-rich strand. These G-overhangs function as substrates for telomerase-mediated strand extension, and are critical for end-protection of telomeres. These functions and their regulations are mediated by specific G-overhang binding proteins. In species of the plant order Asparagales, telomere motifs have diverged from a type typical of the plant Arabidopsis thaliana (TTTAGGG)(n) to a type typical of human (TTAGGG)(n). Presumably, this change in motif had an impact on the structure of the telomere and/or the binding of telomeric proteins, including the G-overhang binding proteins. Therefore, we analyse here nucleoprotein complexes formed by protein extracts from plants possessing human-type telomeres (Muscari armeniacum and Scilla peruviana). Proteins were characterized that bind to the G-rich strand of both telomere motifs, or to the ancestral Arabidopsis-type motif alone, but none bound to double-stranded or C-rich complementary strand telomere motifs. We demonstrate the size, sequence-specificity and thermostability of these DNA-binding proteins. We also analysed the formation of complexes from renatured protein fractions after SDS-PAGE (sodium-dodecyl-sulphate polyacrylamide-gel-electrophoresis). We discuss the evolutionary consequences of protein binding flexibility, to act on both ancestral and present telomeric sequences. Of particular interest is that the ancestral repeat, which is thought not to form the telomere, binds the proteins most strongly. These data are discussed in line with other known plant telomere-binding proteins and with the complex nature of the telomere in Asparagales carrying a human-type motif.     

Formation of the G-quadruplex and i-motif structures in retinoblastoma susceptibility genes (Rb)

The formation of G-quadruplex and i-motif structures in the 5′ end of the retinoblastoma (Rb) gene was examined using chemical modifications, circular dichroism (CD) and fluorescence spectroscopy. It was found that substitutions of 8-methylguanine at positions that show syn conformations in antiparallel G-quadruplexes stabilize the structure in the G-rich strand. The complementary C-rich 18mer forms an i-motif structure, as suggested by CD spectroscopy. Based on the C to T mutation experiments, C bases participated in the C–C⁺ base pair of the i-motif structure were determined. Experiments of 2-aminopurine (2-AP) substitution reveal that an increase of fluorescence in the G-quadruplex relative to duplex is attributed to unstacked 2-AP within the loop of G-quadruplex. The fluorescence experiments suggest that formation of the G-quadruplex and i-motif can compete with duplex formation. Furthermore, a polymerase arrest assay indicated that formation the G-quadruplex structure in the Rb gene acts as a barrier in DNA synthesis.     

The protein profile of acetazolamide-treated sera in mice bearing Lewis neoplasm

The aim of the present research is to analyze the proteome of neoplasm serum before and after treated with acetazolamide (20, 40, 80 mg kg(-1) d(-1) for 3 days p.o.). The Lewis lung carcinoma mice were used and carried out a comprehensive proteomic analysis by using the technologies of high-resolution two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and mass spectrometry (MS). The results showed that the acetazolamide could dramatically reduce the lung metastasis and primary tumor growth. Its most potent inhibition rate on lung metastases was reach to 77.7% at the dose of 80 mg kg(-1) d(-1). The two dimension electrophoresis and software analysis reveal 393 protein spots in control gel, 385 protein spots were detected in treated gel and matched 209 protein spots with control gel, indicating that intensive changes had occurred during the process of treatment. Two obviously different spots were cut off from gel and for the peptide mass fingerprinting. Data base searching showed the two proteins' peptide much more mach with Histone H2B fragment and Ubc-like protein CROC1 fragment. The results suggest that acetazolamide has a strong anti-tumor and anti-metastasis effect on Lewis-lung-carcinoma. The mechanism may be related to its regulation on plenty of proteins, in particular, on upregulation of H2B and CROC-1 expression of postreplicational DNA repair related protein in serum.     

The PNA-DNA hybrid I-motif: implications for sugar-sugar contacts in i-motif tetramerization

We have created a hybrid i-motif composed of two DNA and two peptide nucleic acid (PNA) strands from an equimolar mixture of a C-rich DNA and analogous PNA sequence. Nano-electrospray ionization mass spectrometry confirmed the formation of a tetrameric species, composed of PNA–DNA heteroduplexes. Thermal denaturation and CD experiments revealed that the structure was held together by C-H⁺-C base pairs. High resolution NMR spectroscopy confirmed that PNA and DNA form a unique complex comprising five C-H⁺-C base pairs per heteroduplex. The imino protons are protected from D₂O exchange suggesting intercalation of the heteroduplexes as seen in DNA₄ i-motifs. FRET established the relative DNA and PNA strand polarities in the hybrid. The DNA strands were arranged antiparallel with respect to one another. The same topology was observed for PNA strands. Fluorescence quenching revealed that both PNA–DNA parallel heteroduplexes are intercalated, such that both DNA strands occupy one of the narrow grooves. H1′–H1′ NOEs show that both heteroduplexes are fully intercalated and that both DNA strands are disposed towards a narrow groove, invoking sugar–sugar interactions as seen in DNA₄ i-motifs. The hybrid i-motif shows enhanced thermal stability, intermediate pH dependence and forms at relatively low concentrations making it an ideal nanoscale structural element for pH-based molecular switches. It also serves as a good model system to assess the contribution of sugar–sugar contacts in i-motif tetramerization.     

Small circular DNAs for synthesis of the human telomere repeat: varied sizes, structures and telomere-encoding activities

We describe the construction, structural properties and enzymatic substrate abilities of a series of circular DNA oligonucleotides that are entirely composed of the C-rich human telomere repeat, (CCCTAA)_n. The nanometer-sized circles range in length from 36 to 60 nt, and act as templates for synthesis of human telomere repeats in vitro. The circles were constructed successfully by the application of a recently developed adenine-protection strategy, which allows for cyclization/ligation with T4 DNA ligase. Thermal denaturation studies showed that at pH 5.0, all five circles form folded structures with similar stability, while at pH 7.0 no melting transitions were seen. Circular dichroism spectra at the two pH conditions showed evidence for i-motif structures at the lower pH value. The series was tested as rolling circle templates for a number of DNA polymerases at pH = 7.3–8.5, using 18mer telomeric primers. Results showed that surprisingly small circles were active, although the optimum size varied from enzyme to enzyme. Telomeric repeats 1000 nt in length could be synthesized in 1 h by the Klenow (exo-) DNA polymerase. The results establish a convenient way to make long human telomeric repeats for in vitro study of their folding and interactions, and establish optimum molecules for carrying this out.