The single-stranded (TTAGGG)n tail of human telomeric DNA is known to form stable G-quadruplex structures. Optimal telomerase activity requires the nonfolded single-stranded form of the primer, and stabilization of the G-quadruplex form is known to interfere with telomerase binding. We have identified 3,4,9, 10-perylenetetracarboxylic diimide-based ligands as potent inhibitors of human telomerase by using a primer extension assay that does not use PCR-based amplification of the telomerase primer extension products. A set of NMR titrations of the ligand into solutions of G-quadruplexes using various oligonucleotides related to human telomeric DNA showed strong and specific binding of the ligand to the G-quadruplex. The exchange rate between bound and free DNA forms is slow on the NMR time scale and allows the unequivocal determination of the binding site and mode of binding. In the case of the 5'-TTAGGG sequence, the ligand-DNA complex consists of two quadruplexes oriented in a tail-to-tail manner with the ligand sandwiched between terminal G4 planes. Longer telomeric sequences, such as TTAGGGTT, TTAGGGTTA, and TAGGGTTA, form 1:1 ligand-quadruplex complexes with the ligand bound at the GT step by a threading intercalation mode. On the basis of 2D NOESY data, a model of the latter complex has been derived that is consistent with the available experimental data. The determination of the solution structure of this telomerase inhibitor bound to telomeric quadruplex DNA should help in the design of new anticancer agents with a unique and novel mechanism of action.

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A major control element of the human c-myc oncogene is the nuclease-hypersensitive purine/pyrimidine-rich sequence. This double-stranded DNA fragment, corresponding to the 27-base pair segment in the nucleasehypersensitive element of the c-myc promoter region, forms a stable Watson-Crick double helix under physiological conditions. However, this duplex DNA can be effectively converted to G-quadruplex DNA by a small molecular weight ligand. Both intermolecular and intramolecular G-quadruplex forms can be induced by this ligand. Similar transitional changes are also observed with the duplex telomeric sequence from the Oxytricha species. These results provide additional support to the idea that G-quadruplex structures may play structural roles in vivo and also provide insight into novel methodologies for rational drug design. These structurally altered DNA elements might serve as regulatory signals in gene expression or in telomere dynamics and hence are promising targets for drug action.The protein product of the c-myc protooncogene plays a vital role in the process of cellular growth and differentiation (1). Deregulation of c-myc expression has been detected in many cancers and is believed to be an important step in tumorigenesis (2). The control of c-myc gene expression is a complex process and occurs at various steps of transcription, such as initiation, elongation, and attenuation, as well as during the post-transcriptional stages. Although the mechanisms involved in this regulation are not yet completely understood, a major control element of the human c-myc oncogene has been localized. This is a purine/pyrimidine-rich region located 115 bases upstream from the P1 promoter, which controls up to 95% of the total c-myc transcription (3, 4). This DNA segment is highly sensitive to DNase I and S1 nuclease (5, 6) and is termed the nuclease-hypersensitive element (NHE).1 The appearance of this hypersensitive site is coupled with transcription activation of the c-myc oncogene. Structural variations in the NHE can influence the binding of transcription factors. Transcription factors such as heterogenous nuclear ribonucleoprotein K and nucleoside diphosphate kinase B (7) bind sequence specifically to the pyrimidine-rich strand of the NHE and activate c-myc transcription (8). The transacting factors heterogenous nuclear ribonucleoprotein A/B (9) and cellular nucleic acid-binding protein (10) bind to the NHE and are shown to augment c-myc expression in vitro. Apart from protein factors, antisense oligonucleotides bind to the NHE and repress c-myc transcription in vitro (11,12). Formation of a colinear triplex between the synthetic oligonucleotide and the NHE was proposed to cause this observed repression in transcription.The NHE has a high potential to form atypical DNA structures under superhelical stress. It was proposed to be in a slow equilibrium between a Watson-Crick base-paired double helix and an atypical DNA structure (6). Many models have been suggested to explain the conformational changes observed in the NHE...

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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.

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WW domains mediate protein recognition, usually though binding to proline-rich sequences. In many proteins, WW domains occur in tandem arrays. Whether or how individual domains within such arrays cooperate to recognize biological partners is, as yet, poorly characterized. An important question is whether functional diversity of different WW domain proteins is reflected in the structural organization and ligand interaction mechanisms of their multiple domains. We have determined the solution structure and dynamics of a pair of WW domains (WW3-4) from a Drosophila Nedd4 family protein called Suppressor of deltex (Su(dx)), a regulator of Notch receptor signaling. We find that the binding of a type 1 PPPY ligand to WW3 stabilizes the structure with effects propagating to the WW4 domain, a domain that is not active for ligand binding. Both WW domains adopt the characteristic triple-stranded ␤-sheet structure, and significantly, this is the first example of a WW domain structure to include a domain (WW4) lacking the second conserved Trp (replaced by Phe). The domains are connected by a flexible linker, which allows a hingelike motion of domains that may be important for the recognition of functionally relevant targets. Our results contrast markedly with those of the only previously determined three-dimensional structure of tandem WW domains, that of the rigidly oriented WW domain pair from the RNA-splicing factor Prp40. Our data illustrate that arrays of WW domains can exhibit a variety of higher order structures and ligand interaction mechanisms.WW domains are small protein interaction modules found in a wide range of eukaryotic signaling and structural proteins (1). The domain is a small three-stranded ␤-sheet stabilized by the stacking of several conserved aromatic and proline residues (2). Differences in residue identity at the binding surface result in a variation in ligand specificity that is used as the basis to divide WW domains into groups. For example, in group I WW domains that bind PPXY sequences (3), the Tyr is a key specificity residue and is accommodated by a largely hydrophobic pocket on the concave binding surface consisting of conserved Ile (or Val/Leu), His, and Gln (or Arg/Lys) residues. The Pro residues of the ligand contribute to the binding by stacking against the Trp and Tyr residues that form a second interaction site (4). It is evident that, since a number of proteins are likely to contain WW domain recognition sites, further factors most probably contribute to increasing affinity and specificity of a WW domain for a target. For example, the Pro-rich sequence in ␤-dystroglycan targeted by the dystrophin WW domain requires a composite binding surface provided by the WW domain and an adjacent EF hand (4). The binding of murine Nedd4 to the amiloride-sensitive epithelial sodium channel requires direct involvement of two of its three WW domains (5). Indeed, WW domains often exist in multiple numbers within a protein. Multiple modules may act in concert to achieve greater specificity for a target ...

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