DNA oligomers, listed in Table 1, were supplied by Operon Technologies. The oligonucleotides were purified by electrophoresis through a denaturing gel of 12% polyacrylamide (acrylamide:bisacrylamide, 19:1), 8 M urea as described (³³), except that salt and acrylamide residues were removed by twice precipitating the DNA with ethanol.

Purified DNA oligomers that were 5? end-labeled with ³²P in a T4 polynucleotide kinase catalyzed reaction (³⁴) were maintained in their single-stranded conformation by being stored as a solution of 0.25 ?M DNA in water and boiled immediately prior to use. Bimolecular quadruplex structures of ³²P-5?-labeled d(CGG)_n oligomers with or without interspersed d(AGG) triplets were formed by incubating at 55?C for 20?24 h a single oligomer or an indicated mixture of two oligomers (~50 ?M total DNA concentration) in 10 ?l TE buffer (10 mM Tris?HCl buffer, pH?8.0, 1 mM EDTA) containing 500 mM NaCl. The reaction was terminated by rapidly cooling and diluting the mixtures 10-fold in 25 mM Tris?HCl buffer pH 8.0, 1 mM EDTA, 0.5?mM DTT and 20% glycerol. To resolve slowly migrating tetraplex DNA complexes from remaining single-stranded oligomers, DNA was electrophoresed under 80?100 V and at 4?C through a non-denaturing 8?12% polyacrylamide gel (acrylamide:bisacrylamide, 30:1.2) in 0.5? TBE buffer (1.25 mM EDTA in 45 mM Tris?borate buffer, pH 8.3) containing 50?mM NaCl. Concentrations of labeled quadruplex forms of the various oligomers were deduced from measurements of the specific radioactivity of source oligomers and of the radioactivity of the electrophoretically-separated tetraplex structures.

DNA helicase reaction mixtures contained in a final volume of 10 ?l: 40 mM Tris?HCl buffer, pH 7.4, 4 mM MgCl₂, 20 mM KCl, 5 mM DTT, 1 mM ATP, 10 ?g BSA and 300 fmol ³²P-5?-labeled tetraplex DNA. After adding specified amounts of recombinant Werner DNA helicase (WRN) purified to homogeneity (³⁵), the reaction mixtures were incubated at 37?C for indicated time periods. Reactions were terminated by rapidly cooling the samples and adding 2 ?l of a solution of 40% glycerol, 50 mM EDTA, 2% SDS, 3% bromophenol blue and 3% xylene cyanol. Displaced DNA single-strands were resolved from remaining tetraplex DNA by electrophoresis at 4?C and under 80?120 V through a non-denaturing 8?12% polyacrylamide gel in 0.5? TBE buffer, 10 mM NaCl. Gels that were dried on Whatman 3 filter paper were exposed to autoradiographic film to visualize resolved DNA bands. Amounts of tetraplex DNA and of displaced single-strands were quantified by exposing gels dried on Whatman DE81 filter paper to phosphorimager plate (Fuji).

To study the effect of interspersed d(AGG) triplets on structural transformations of d(CGG)_n runs, we first inquired whether d(AGG)-containing d(CGG)_n oligonucleotides formed bimolecular tetraplex structures. ³²P-5?-labeled d(CGG)₇, tail d(CGG)₁₈ and tail d(A/CGG)₁₈ 1:7 [the 1:7 ratio refers to the frequency of interspersed d(AGG) triplets within the d(CGG)_n run; see Table 1 for the nucleotide sequence of all oligomers used] were incubated at 55?C for 20 h in the presence of 500 mM NaCl. As seen in Figure 1, all three oligomers formed electrophoretically-retarded DNA structures that could be heat denatured into their rapidly migrating single-strand constituents. Similar complexes were also formed by d(CGG)₁₈ and d(A/CGG)₁₈ 1:5 under the described incubation conditions. Additionally, complexes of d(CGG)₁₈ and d(A/CGG)₁₈ 1:5 or of their 3?-tailed counterparts did not form in the absence of Na⁺ ions (results not shown). The above results indicated that the presence of the non-d(CGG) unpaired 3?-tail did not affect complex formation. A non-d(CGG) 3? single-strand tail was required for the unwinding of tetraplex DNA by WRN helicase (³⁶). Since WRN was used at a later stage of this study (vide infra), tailed oligomers were used throughout this work. The observed alkali ion-dependence of complex formation suggested that complexes of d(CGG)₁₈ and d(A/CGG)₁₈ 1:5 with or without an unpaired tail represented tetraplex structures of the oligomers (²⁶?²⁸). To determine the stoichiometry of the observed retarded DNA structures, ³²P-5?-labeled d(CGG)₇ was incubated at 55?C in the presence of Na⁺ with equimolar amounts of tail d(CGG)₁₈ or tail d(A/CGG)₁₈ 1:7. As shown in Figure 1, in addition to retarded complexes characteristic of each oligomer, the DNA mixtures yielded third hybrid species. As previously described (²⁸,³⁶,³⁷), generation of three retarded bands in mixtures of d(CGG)₇ with tail d(CGG)₁₈ or tail d(A/CGG)₁₈ 1:7 indicated that the slowly migrating complexes of tail d(CGG)₁₈ with or without interspersed d(AAG) triplets represented G?2 bimolecular DNA structures. That these DNA complexes were tetrahelices was indicated by the obligatory requirement for alkali cations for their formation and stability (results not shown; ²⁶,³⁶). More specifically, we have shown that similar complexes of d(CGG)₇ displayed circular dichroism (CD) spectrum characteristic of antiparallel tetraplex structures (³⁷). By probing the guanine N7 position with dimethylsulfate we also showed that these structures were maintained by non-Watson?Crick guanine?guanine hydrogen bonds (²⁶). The antiparallel tetraplex structure of oligo d(CGG)_n was also demonstrated by NMR analysis (²⁸). The indistinguishable electrophoretic behavior, stoichiometry and obligatory requirement for alkali ions of G?2 tetraplex tail d(CGG)₁₈ and of the retarded complex of tail d(A/CGG) 1:7 suggested that the latter also represented a G?2 tetraplex structure.

We first studied the effect of interspersed d(AGG) triplets on the extent of formation of G?2 tetraplex structures of d(CGG)_n. Increasing concentrations of 5?-³²P-labeled tail d(CGG)₁₈ and of oligomers that contained interspersed triplets at d(AGG) to d(CGG) frequencies of 1:5, 1:7 or 1:10, were incubated at 55?C and in the presence of 500 mM NaCl. Formed tetraplex DNA complexes were resolved electrophoretically from remaining single-strands and their proportions were determined by phosphorimager analysis. As seen in Figure 2A, G?2 tail d(CGG)₁₈ tetraplex was progressively accumulated in a second-order reaction as previously demonstrated for shorter d(CGG)_n tracts (²⁶,³⁵,³⁶). However, whereas up to ~30% of ~35 ?M tail d(CGG)₁₈ accumulated in a G?2 tetraplex form, only 2.5?8% of >40 mM of oligomers that contained interspersed d(AGG) triplets were found in G?2 tetraplex forms (Fig. 2A).

To compare the thermal stabilities of G?2 tetraplex structures of monotonous and d(AGG)-containing d(CGG)₁₈ oligomers, they were heated at 53?C for increasing time periods and the amounts of denatured tetraplex structures were quantified by phosphorimager analysis. As seen in Figure 2B, whereas G?2 tail d(CGG)₁₈ remained largely intact after up to 15 min at 53?C, ~35 and 50% of G?2 tail d(A/CGG)₁₈ 1:7 and 1:10, respectively, were denatured under the same conditions. The greater thermal lability of d(AGG)-containing d(CGG)₁₈ tetraplex structures was confirmed by direct measurements of their melting temperatures. Whereas the T_m value of G?2 tail d(CGG)₁₈ was 57?C, the T_m of G?2 tail d(A/CGG)₁₈ 1:7 was 48.5?C (Fig. 3). Similarly, G?2 tail d(A/CGG)₁₈ 1:10 was found to have a T_m value of 48?C (results not shown). Notably, formation or denaturation of the tetraplex DNA structures were critically affected by the ionic strength of the reaction mixture. Thus, whereas the presence of 500 mM NaCl allowed formation at 55?C of tetraplex structures of the oligomers (Figs 1 and 2), these tetrahelical structures were denatured in the presence of 50?mM NaCl at 48?57?C (Fig. 3).

We have shown recently that human WRN DNA helicase unwinds bimolecular G?2 tetraplex forms of d(CGG)₇ and that this activity required unpaired non-d(CGG) tails at the 3? or 5? end of the oligomer (³⁶). We inquired, therefore, whether the decreased heat stability of d(AGG)-containing tetraplex structures of d(CGG)₁₈ is also reflected in increased unwinding by WRN helicase. Similar amounts of 5?-³²P-labeled G?2 tail d(CGG)₁₈, G?2 tail d(A/CGG)₁₈ 1:7 or G?2 tail d(A/CGG)₁₈ 1:10 were incubated under helicase reaction conditions with increasing amounts of WRN. Destabilization of the tetraplex DNA structures was assessed by non-denaturing gel electrophoresis DNA strand displacement analysis (see Materials and Methods). As seen in Figure 4A, whereas G?2 tail d(CGG)₁₈ was only minimally unwound by WRN under the helicase assay conditions, large proportions of d(A/CGG)₁₈ 1:7 or G?2 d(A/CGG)₁₈ 1:10 were destabilized by the enzyme. Quantitative results of a typical experiment, shown in Figure 4B, indicated that whereas G?2 tail d(CGG)₁₈ was unwound to a maximum extent of 25%, up to ~60 and ~80% of G?2 tetraplex forms of tail d(A/CGG)₁₈ 1:7 or tail d(A/CGG)₁₈ 1:10, respectively, became destabilized. Kinetic analysis confirmed that the presence of interspersed d(AGG) triplets accelerated the unwinding of G?2 tetraplex d(CGG)₁₈ by WRN. Reaction mixtures, each containing 300 fmol 5?-³²P-labeled tail d(CGG)₁₈, tail d(A/CGG)₁₈ 1:7 or tail d(A/CGG)₁₈ 1:10, were incubated for increasing time periods with 75 fmol WRN under helicase assay conditions. Electrophoretic resolution of intact and unwound tetraplex DNA shown in Figure 5A indicated that whereas G?2 tail d(CGG)₁₈ was unwound to a limited extent over the duration of WRN action, substantial proportions of G?2 tail d(A/CGG)₁₈ 1:7 or G?2 tail d(A/CGG)₁₈ 1:10 were dissociated into their single-strand constituents. Quantitative analysis of the results of a typical experiment, presented in Figure 5B, indicated that the initial rates of WRN-catalyzed unwinding of G?2 tail d(A/CGG)₁₈ 1:7 or G?2 tail d(A/CGG)₁₈ 1:10 were higher than for tail d(CGG)₁₈. Also, by 30 min, WRN destabilized ~60 and ~85% of G?2 tail d(A/CGG)₁₈ 1:7 and G?2 tail d(A/CGG)₁₈ 1:10, respectively, whereas only ~25% of G?2 tail d(CGG)₁₈ became unwound.

This work was initiated in the laboratory of Dr Lawrence A. Loeb at the University of Washington, Seattle. M.F. wishes to thank L.A.L. for his long standing support and friendship. The work was supported by grants to M.F. from the Israel Science Foundation, the US?Israel Binational Fund, the Conquer Fragile X Inc. Foundation and by a Technion V.P.R. Fund, Charles Krown Research Fund and by NCI grant P01-CA-47852 to L.A.L.