The solution structure of the DNA quadruplex formed by the association of two strands of the DNA oligonucleotide, d(G3T4G3), in NaCl solution has been determined by 1H two‐dimensional NMR techniques, full relaxation matrix calculations and restrained molecular dynamics. The refined structure incorporates the sequences 5′‐GlsG2AG3AT4AT5AT6AT7AG8sG9AG10A‐3′ and 5′‐G11sG12AG13AT14AT15A. T16AT17AG18sG19sG20A‐3′ (where S and A denote syn and anti, respectively) in a three‐quartet, diagonal‐looped structure that we [Strahan, G. D., Shafer, R. H. & Keniry, M. A. (1994) Nucleic Acids Res. 22, 5447–5455] and others [Smith, F. W., Lau, F. W. & Feigon, J. (1994) Proc. Natl Acad. Sci. USA 91, 10546–10550] have described. The loop structure is compact and incorporates many of the features found in duplex hairpin loops including base stacking, intraloop hydrogen bonding and extensive van der Waals' interactions. The first and third loop thymines stack over the outermost G‐quartet and are also associated by hydrogen bonding. The second and the fourth loop thymines fold inwards in order to enhance van der Waals' interactions. The unexpected sequential syn‐syn deoxyguanosines in the quadruplex stem appear to be a direct consequence of the way DNA oligonucleotides fold and the subsequent search for the most stable loop structure. The implications of loop sequence and length on the structure of quadruplexes are discussed.

The catalytic core of Escherichia coli DNA polymerase III contains three tightly associated subunits, the ␣, , and subunits. The subunit is the smallest and least understood subunit. The three-dimensional structure of in a complex with the unlabeled N-terminal domain of the subunit, 186, was determined by multidimensional nuclear magnetic resonance spectroscopy. The structure was refined using pseudocontact shifts that resulted from inserting a lanthanide ion (Dy 3؉ , Er 3؉ , or Ho 3؉ ) at the active site of 186. The structure determination revealed a three-helix bundle fold that is similar to the solution structures of in a methanolwater buffer and of the bacteriophage P1 homolog, HOT, in aqueous buffer. Conserved nuclear Overhauser enhancement (NOE) patterns obtained for free and complexed show that most of the structure changes little upon complex formation. Discrepancies with respect to a previously published structure of free (Keniry et al., Protein Sci. 9:721-733, 2000) were attributed to errors in the latter structure. The present structure satisfies the pseudocontact shifts better than either the structure of in methanol-water buffer or the structure of HOT. satisfies these shifts. The epitope of 186 on was mapped by NOE difference spectroscopy and was found to involve helix 1 and the C-terminal part of helix 3. The pseudocontact shifts indicated that the helices of are located about 15 Å or farther from the lanthanide ion in the active site of 186, in agreement with the extensive biochemical data for the -system.The holoenzyme DNA polymerase III (Pol III) is the main replicative polymerase in Escherichia coli (37). It is a remarkable multisubunit enzyme that is capable of extraordinary speed and fidelity of action. Pol III is composed of 10 different subunits, 7 of which act as accessory subunits for a catalytic core composed of 3 tightly bound subunits. The polymerase active site is in the large ␣ subunit (130 kDa) of the core (33, 34). The 3Ј-5Ј proofreading exonuclease activity is located in the ε subunit (28 kDa) (48), specifically, in the N-terminal domain (ε186) (21 kDa), which also contains the binding site of the subunit (9 kDa). The subunit has no unambiguously designated function. The three subunits are arranged linearly such that the ε subunit occupies the central position, binding to both ␣ and (51).The possibility of understanding the detailed function of the Pol III holoenzyme has provoked interest in the structure of this enzyme since its discovery more than 25 years ago. Unfortunately, neither the complete holoenzyme complex nor the catalytic core has been crystallized yet. Instead, there has been considerable effort to solve the structures of individual subunits by both X-ray crystallographic and nuclear magnetic resonance (NMR) methods, and there has been substantial success recently for two of the three elements of the core. The structure of ε186 has been determined by X-ray crystallography (20) and has been modeled from NMR data (8). This structure led to a detailed understanding of ...

The loop of four thymines in the sodium form of the dimeric folded quadruplex [d(G3T4G3)]2 assumes a well-defined structure in which hydrogen bonding between the thymine bases appears to contribute to the stability and final conformation of the quadruplex. We have investigated the importance of the loop interactions by systematically replacing each thymine in the loop with a cytosine. The quadruplexes formed by d(G3CT3G3), d(G3TCT2G3), d(G3T2CTG3) and d(G3T3CG3) in the presence of 150 mM Na+ were studied by gel mobility, circular dichroism and 1H NMR spectroscopy. The major species formed by d(G3CT3G3), d(G3TCT2G3) and d(G3T3CG3) at 1 mM strand concentration at neutral pH is a dimeric folded quadruplex. d(G3T2CTG3) has anomalous behaviour and associates into a greater percentage of linear four-stranded quadruplex than the other three oligonucleotides at neutral pH and at the same concentration. The linear four-stranded quadruplex has a greater tendency to oligomerize to larger ill-defined structures, as demonstrated by broad 1H NMR resonances. At pH 4, when the cytosine is protonated, there is a greater tendency for each of the oligonucleotides to form some four-stranded linear quadruplex, except for d(G3T2CTG3), which has the reverse tendency. The experimental results are discussed in terms of hydrogen bonding within the thymine loop.

The preferred residence sites and the conformation of DNA-bound polyamines are central to understanding the regulatory roles of polyamines. To this end, we have used a series of selective (13)C-edited and selective total correlation spectroscopy-edited one-dimensional (1D) nuclear Overhauser effect spectroscopy NMR experiments to determine a number of intramolecular (1)H nuclear Overhauser effect (NOE) connectivities in (13)C-labelled spermine bound to the thrombin-binding aptamer. The results provide evidence that the aptamer-bound spermine adopts a conformation that optimizes electrostatic and hydrogen bond contacts with the aptamer backbone. The distance between the nitrogen atoms of the central aminobutyl is reduced by an increase in the population of gauche conformers at the C6-C7 bonds, which results in either a curved or S-shaped spermine conformation. Molecular modelling contributes insight toward the mode of spermine binding of these spermine structures within the narrow grooves of DNA quadruplexes. In each case, the N5 ammonium group makes hydrogen bonds with two nearby phosphates across the narrow groove. Our results have implications for the understanding of chromatin structure and the rational design of quadruplex-binding drugs.

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