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Nucleic Acids Research, 2002, Vol. 30, No. 23 5276-5283
© 2002 Oxford University Press

G-quartets assembly within a G-rich DNA flap. A possible event at the center of the HIV-1 genome

Sébastien Lyonnais, Candide Hounsou1, Marie-Paule Teulade-Fichou1, Josette Jeusset, Eric Le Cam and Gilles Mirambeau*

Laboratoire de Microscopie Moléculaire et Cellulaire, CNRS UMR 8126, Institut Gustave Roussy, 94805 Villejuif, France and 1 Laboratoire de Chimie des Interactions Moléculaires, Collège de France, UPR 285, 11 Place Marcelin Berthelot, 75005 Paris, France

*To whom correspondence should be addressed. Tel: +33 1 42114880; Fax: +33 1 42115494; Email: mirambe{at}igr.fr

Received June 28, 2002; Revised and Accepted September 28, 2002


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Stretches of guanines can associate in vitro through Hoogsteen hydrogen bonding to form four-stranded structures. In the HIV-1 central DNA flap, generated by reverse transcriptase at the end of retrotranscription, both the two 99 nt-long overlapping (+) strands contain two adjacent tracts of guanines. This study demonstrates that oligonucleotides containing these G-clusters form highly stable G-quadruplexes of various structures in vitro, whose formation was controlled by an easy and reversible protocol using sodium hydroxide. Among these sequences, a G'2 hairpin dimer was the most stable structure adopted by the 5'-tail of the (+) downstream strand. Since the two (+) strands of the HIV-1 central DNA flap hold these G-clusters, and based on the properties of reverse branch migration in DNA flaps, constructions using HIV-1 sequences were assembled to mimic small DNA flaps where the G-clusters are neighbors. G-quartets were successfully probed in such flaps. They were induced by potassium and by a dibenzophenanthroline derivative already known to stabilize them. Such results suggest some function(s) for G-quartets associated with a DNA flap in the HIV-1 pre-integration steps, and argue for their transient formation during the processing of G-rich DNA flaps at the time of replication and/or repair.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
G-quadruplex structures are formed by nucleic acids containing runs of guanines arranged in stacked G-quartets maintained by Hoogsteen base pairing (1–3). Interest in G-DNA has increased since the discovery that telomeric DNA sequences are able to adopt intramolecular quadruplex structures in vitro, which can inhibit telomere elongation by telomerase under physiological conditions (4). Quadru plex conformations of telomeres are thus of considerable interest for designing new anticancer drugs (4). G-quartet structures have been shown to form in distinct genomic microenvironments in vitro, such as the c-myc promoter (5) and the fragile X-syndrome triplet repeats (6), and were subsequently described as targets for several proteins, including Ku autoantigen (7), the ß-subunit of telomere-binding protein from Oxytricha (8), topoisomerase I (9) and hnRNP A1 (10). This strongly suggests that G-quadruplexes can be formed occasionally in genomic DNA. An interesting hypothesis is that G-quartets may occur during DNA replication and interfere with the progression of the replication fork, leading to genomic instability, replication defects or aberrant recombination (11). Effectively, it has been shown that RecQ-type helicases such as the Werner’s syndrome helicase (WRN), whose defect in Werner’s syndrome causes premature aging and chromosomal instability, can unwind G-DNA (12) and alleviate the pausing of yeast polymerase {delta} at sites of G-quadruplexes (13).

G-quadruplexes are of remarkable stability and have a preferential affinity for monovalent cations (typically K+ or Na+), which exhibit a suitable size for interacting with the electronegative carbonyl oxygen ring inside the G-quartet (14,15). Quadruplex DNA has been reported to be highly polymorphic, three major conformations being commonly found: G4-DNA, in which the four sugar backbones run in a parallel orientation; G'2-DNA anti-parallel hairpin dimers; and intramolecular folded species designed G4'-DNA (15). Higher molecular weight species, the G-wires, can also be formed by non-aligned oligonucleotides containing two or more G-repeats (16). The transitions between the various conformations could be modulated by monovalent cations, time, length, sequence and concentration of the nucleic acids considered.

After virus entry into the host cell, lentiviral replication requires generation of a subviral particle termed the pre-integration complex (PIC), composed of the retrotranscribed DNA and at least two major viral proteins, integrase and Vpr, and low amounts of MAp, NCp and reverse transcriptase (RT) (17–19). This HIV-1 PIC is actively imported into the host nucleus by a rather undefined mechanism (20). The full-length product of HIV-1 reverse transcription is a double-stranded DNA producing at its centre a 99 nt single-stranded tail of the (+) strand, the central DNA flap (Fig. 1) (21). This unusual structure is generated when RT-mediated synthesis of the 3'-polypurine tract (PPT)-primed (+) strand displaces 99 nt along the pre-existing (+) strand elongated from the central PPT (cPPT) primer (21,22). Processing of the central DNA flap was shown to act as a cis-determinant element in HIV-1 DNA nuclear import (23) and, when inserted into HIV-1-derived lentiviral vectors, to confer a striking increase in gene transduction in target cells (24,25). However, its precise implication is still unclear (26).



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  		Figure 1. (A) Final DNA product of HIV-1 retrotranscription containing its 99 nt-long central DNA flap and the two LTR repeats at both ends, according to the processing of HIV-1 (+) strand DNA synthesis (21,22). (B) Representation of the HIV-1 central DNA flap and localization of the ODNs used in this article. (+)U, (+) upstream strand; (+)D, (+) downstream strand.

 
The following results present the ability of HIV-1 central DNA flap (+) strand-derived oligodeoxynucleotides (ODN) containing a G6-G4 cluster to associate into a variety of DNA quadruplexes and, moreover, to interact via G-quartets in flap structures reconstituted in vitro.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Oligonucleotides were purchased from MWG-Biotech. Sequences, names and relative positions in the HIV-1 central DNA flap (Bru isolate) are given in Table 1 and Figure 1B. Monometaquinacridine3 (MMQ3) (see Fig. 6D) was synthesized as previously described (27).


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  		Table 1. HIV-1 central flap oligonucleotides and sequence motifs (5'->3')

 


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  		Figure 6. MMQ3 promotes the formation of F' species. Reactions were performed with F1 and F2 incubated for 20 h at 52°C. (A) F1 construction. Lane 1, mutODN(–)/ODN3 duplex; lane 2, control F1; lanes 3–6, F1 incubated in 100 mM KCl in the absence (lane 3) or presence of 2, 5 and 10 µM MMQ3. (B) F2 construction. Lane 1, ODN(–)/ODN3 duplex, Other lane compositions were identical to the preceding experiment. (C) Fluorimaging quantification of F'1 and F'2 formation by increasing the amounts of MMQ3, as the ratio between F' species and the sum of F + F'. (D) Monometaquinacridine3 (MMQ3).

 
G-quartet formation
Quadruplexes were formed by interstrand association of ODNs as indicated in the figure legends. For Figures 1, 3 and 4, ODNs were incubated at 20–30 µM in TE (10 mM Tris–HCl, pH 7.5, 1 mM EDTA) with the indicated salts, diluted 1:5 with 50 mM salt (KCl, NaCl or LiCl), 3% glycerol and marker dyes. Before purification, ODN1 oligomer T (ODN1 G4 DNA) was formed with ODN1 (30 µM) incubated in 200 mM KCl/TE at 40°C for 10 h, and ODN2 oligomer D (ODN2 G'2 DNA) was formed using ODN2 (30 µM) incubated in 250 mM KCl/TE at 35°C for 45 h. Samples were then electrophoresed on 8% non-denaturing minigels containing 20 mM KCl. Bands corresponding to T and D were identified according to their gel mobility by UV shadowing and excised. DNA was eluted from the crushed gel slices by soaking in 0.5 M potassium acetate, 1 mM EDTA at 37°C for 8–12 h, and purified with an Ultrafree-DA DNA extraction kit coupled to a Microcon 10 (Millipore) according to the manufacturer’s instructions. G-quadruplexes were 5'-end-labeled using [{gamma}-33P]ATP (ICN) and T4 polynucleotide kinase (USB). Labeled substrates were incubated as indicated in the figure legends. Chemical probing using dimethylsulfate (DMS) was strictly performed according to Williamson et al. (3) and loaded on a 20% sequencing gel.



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  		Figure 3. Quadruplexes formed by ODN2 in the presence of monovalent cations. (A) ODN2 and its mutated derived (30 µM) were incubated for 10 h at 50°C and loaded on a 10:16% polyacrylamide gradient gel. Lanes 1–3, ODN2 mutants incubated in 100 mM KCl; lane 4, ODN2 monomer obtained with 50 mM NaOH; lane 5, ODN2 incubated in 150 mM NaCl; lane 6, ODN2 incubated in 100 mM KCl. W, T, X and D indicate the positions of G-quadruplexes, M is that of the unfolded monomer. (B) DMS methylation protection of D. 33P-radiolabeled D was mixed with the indicated amounts of NaOH in 125 mM KCl prior to DMS methylation and cleavage with piperidine.

 


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  		Figure 4. Kinetics of the assembly of ODN2 structures in the presence of monovalent cations. ODN2 (10 µM) was incubated at 40°C for specified times, after 30 mM NaOH denaturation, in the presence of 100 mM KCl (lanes 1–3), 200 mM NaCl (lanes 4–6) or 200 mM LiCl (lanes 7–8). Lane 9, no added salts. Samples were loaded on 16% acrylamide gels and stained with SYBRgreen.

 
Gel electrophoresis
Native gel electrophoresis was performed at room temperature in polyacrylamide gels (29:1 polyacrylamide:bisacrylamide; Promega) or high resolution agarose (Resophor; Eurobio) in 0.5x TBE (0.45 M Tris–borate, pH 8.3, 0.5 mM EDTA) at 8–10 V/cm. DNA staining was performed with a SYBRgreen I & II mix (Molecular Probes), each diluted 1/10 000. Pre-staining was also performed by incubating DNA samples with a 1/5000 SYBRgreen I & II mix at 4°C in the dark and gels were run in a dark room to avoid loss of SYBRgreen fluorescence. In spite of a reduced sensitivity (~10-fold under the gel staining method), pre-staining allowed loss of background fluorescence when it did not affect sample migration. Gels were visualized by either scanning on a fluorimager (Storm; Molecular Dynamics) or UV transillumination at 254 nm and standard Polaroid apparatus.

Denaturation of G4-DNA by NaOH
ODNs were incubated at room temperature for 15 min in fresh NaOH solution. Samples were then neutralized with HCl and adjusted to neutral pH with 20 mM final concentration HEPES, pH 7.5. Equivalence between HCl and NaOH concentrations were fixed after acid–base titration and solutions were always diluted at the same time, using identical volumes. Storage of the NaOH stock solution never exceeded 1 week.

Flap substrates and related G-quartets
Flap substrates were constructed by annealing ODN4 and ODN3, or mutODN3, to templates ODN(–) for F2 construction and mutODN(–) for F1. To avoid undesired G-quartet formation, 7-deaza dG modified bases were used to disrupt the G6 tract corresponding to the cPPT sequence in ODN4. Annealing was carried out in 20 mM HEPES, pH 7.5, 3 mM MgCl2, 0.01% Tween 20 and molar ratios of 1:2:2.7 for ODN(–), ODN3 and ODN4, respectively. Prior to annealing, ODN4 and ODN3 were incubated together for 15 min in 100 mM NaOH, followed by neutralization. Samples were incubated for 15 min at 95°C and for 150 min at 42°C and G-quartet formation was carried out according to the figure legends. Aliquots of 45 ng DNA substrate were pre-stained with SYBRgreen and loaded on 4.5% agarose gels.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Synthetic oligonucleotides derived from the (+) strands of the HIV-1 central DNA flap (Table 1 and Fig. 1B) and containing G-clusters exhibit unusual properties: after incubation for 12 h in 50 mM KCl, followed by native gel electrophoresis, all of them generated slower migrating species or non-migrating oligomers that did not enter the gel (not shown). Since none of these sequences were likely to form classical secondary structures and based on numerous studies on the potassium-driven effect on G-rich DNA (15), we postulated that retarded species could be G-quadruplex structures of various complexities depending on G-runs.

Sodium hydroxide treatment as a tool for G-quartet formation
The structures formed in the presence of K+ were efficiently unfolded by a mild treatment with NaOH, as illustrated in Figure 2A, using the single K+-induced form of ODN1 (T oligomer). In the presence of 100 mM KCl, a condition where G-quartets are strongly stabilized, NaOH addition showed a shift of the fluorescence intensity from T to unfolded monomer (M), which co-migrated with mutODN1 (lane 1), in which two adenines break the G6 tract, and is thus incapable of forming G-quartets. Figure 2B showed the reverse reaction obtained when purified T was treated with 50 mM NaOH and then re-induced with K+. This re-induction of T was not entirely complete, even with concentrated K+ or extended incubation, which we attribute to a limiting DNA concentration for G-quartet formation, due to dilution after the NaOH protocol. T titration by NaOH monitored with SYBRgreen fluorescence showed the efficiency of alkaline treatment and allows the determination of a mid-point transition from T to M at 30 mM NaOH (Fig. 2C). NaOH treatment was also efficient in dissociating the higher order species formed with the other ODNs containing G-runs. Finally, ODN1 conformations in the areas (a), (b) and (c) were probed with DMS (Fig. 2D) in 33P-radiolabeled T stored in 100 mM K+ (lane 1) or treated with 30 or 60 mM NaOH (lanes 2–3). While residues G13 and G15 outside the repeat were sensitive to DMS in all the lanes, G3–G8 were protected against methylation with an inverse relationship to NaOH concentration. The experiments in Figure 2 strongly suggest that T results from the formation of interstrand G-quartets which can be disrupted by NaOH leading to regeneration of the monomer. Because of the presence of a single G-cluster, it can be concluded that T corresponds to a parallel tetraplex (G4), stabilized by six stacked G-quartets.



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  		Figure 2. ODN1 T oligomer is a G4-DNA unfolded by NaOH. (A) Purified T (3 µM) was incubated with the indicated amounts of NaOH according to Materials and Methods in the presence of 100 mM KCl, and loaded on a 20% polyacrylamide minigel. M, unfolded monomer. Lane 1, mutODN1 (30 µM) incubated for 10 h in 200 mM KCl at 35°C. (B) T oligomer (1.5 µM) treated with 50 mM NaOH, incubated for 12 h at 35°C with the indicated amounts of KCl and loaded on a 20% polyacrylamide minigel. Lane 1, purified T control. (C) Denaturation of T by NaOH determined by fluorimaging quantification. (D) DMS methylation protection of T. 33P-radiolabeled T oligomer was methylated by DMS, after (a) no treatment, (b) 30 mM NaOH or (c) 60 mM NaOH treatment, and then cleaved with piperidine.

 
ODN2, the G6-G4 repeats, can adopt either the G4 or G'2 conformation
ODN2 corresponds to the G6-G4 repeats separated by 9 nt in the 5' sequence of the (+)D strand. This ODN was tested for its efficiency to adopt a G-quadruplex conformation. Characterization of the different species was supported by a comparison of the electrophoretic mobilities of modified ODN2 (see Table 1) incubated in K+. Figure 3A shows that modification in the G6 or G4 clusters does not disable G-quartet formation, but constrains these ODNs to parallel associations held by G-quartets, probably at positions 18–21 for A(5–6) ODN2 (lane 1) and at positions 3–8 for A(19–20) ODN2 (lane 2). Co-migration of the bands for A(5–6) and A(19–20) ODN2 indicated that the global shapes of G4-quadruplexes are independent of the position of the G-quartet junction point (3' or 5'). Obviously, the double mutant A(5–6, 19–20) ODN2 was unable to fold into a G-quadruplex (lane 3). In contrast, three bands (T, X and D) appeared when native ODN2 was incubated in K+ (lane 6). In Na+ (lane 5), X, D and especially additional aggregated structures captured in the comb of the gel (W) were observed. According to lanes 1 and 2, the T band can be assigned to the G4-quadruplex, maintained by G-quartets 3' or 5', and the W species to G-wire structures, as already described (15,16). The K+-purified D species were next subjected to DMS chemical probing (Fig. 3B). Lanes 1 and 2 show DMS protection for G18–G21 and G5–G8, while G13, G15 (internal controls) and G3 remained sensitive. Based on its relative mobility versus T and M, and previous studies (1–3), we propose that D is a G'2 quadruplex linked by four stacked G-quartets composed of the G4-tract Hoogsteen base paired with four other guanines in the G6-tract. The conformation of the X species was more difficult to determine since this isomer is rapidly converted into either T or D during purification and storage (not shown).

Potassium strongly favours the fold-back hairpin G'2 for the G6-G4 repeats
Native ODN2 was further incubated in the presence of K+, Na+ or Li+ or without salt for various times (Fig. 4) after NaOH treatment. After 6 h incubation in K+, the predominant structure was the G4 parallel form (T in lane 1) and G-wires (W). T and W, but also X and aggregated species, were found with Na+ (lane 4). At longer incubation times, T disappeared to the benefit of D with K+ (lanes 2–3), whereas in the presence of Na+ T and X were still present at 24 h (lane 5) and were finally converted to D at 48 h (lane 6). We also observed the significant formation of T and W in the absence of salt (lane 9). In contrast, small amounts of T are observed in the presence of Li+ (lanes 7–8). These results suggest that the intermolecular quadruplex is the kinetic structure adopted by the G6-G4 repeats, which is then driven to the thermodynamic product, the G'2-quadruplex, by K+ and to a lesser extent by Na+.

In vitro generation of G-quartets in flap structures simulating the reverse branch migration of the two (+) strands
Efficient reverse branch migration has been proposed for the HIV-1 central DNA flap (22,28), as a consequence of the identical sequences of the two (+) strands being in equilibrium for annealing to the (–) strand. Since these two G6-G4 clusters can promote the formation of the G'2 quadruplex, this prompted us to investigate if G-quartets may occur between the two adjoining (+) strands in the overlap. In that way, the following experiments were performed using a reduced DNA flap built with two strands containing the G6-G4 motif hybridized to the same (–) strand template. Two constructs were made: F1 [ODN3:ODN4:mutODN(–)], where G6-G4 and the immediate surrounding sequences could not anneal to the (–) strand, and F2 [ODN3:ODN4:ODN(–)], where the strands match the native HIV-1 sequence. These substrates were then incubated under different salt conditions and electrophoresed (Fig. 5A and B). When the concentration of K+ was increased, there was increased formation of lower mobility species, F'1 and F'2 (Fig. 5A and B, lanes 5–8). In contrast, without salt (lane 4) or with Li+ (lanes 9–10), no gel shift was observed. As an internal marker, appearance of a band that co-migrated with the K+-induced quadruplex of an ODN4 control incubated alone in the presence of K+ showed the co-formation of a G-quadruplex from the non-annealed ODN4 (Fig. 5A, lanes 5–8). This band was not detected in Figure 5B since it co-migrated with the two-stranded substrates still present in the solution. In Figure 5A and B, lanes 11 and 12 show the flap molecules incubated in 200 mM K+ and 0.005 or 0.02% DMS, respectively. Since N7 of guanine is critical for Hoogsteen base pairing, its methylation should inhibit G-quartet formation. Actually, neither the F'1 or F'2 bands nor the G'2 quadruplex form of ODN4 were observed for 0.02% DMS (lane 12). This indicates that N7 of guanine is essential for the formation of the F'1 and F'2 species. Next, we modified one of the (+) strand in its G6-G4 repeats, to avoid G-quartet formation with itself or with the other (+) strand (Fig. 5C, F3). Incubation of F3 under conditions similar to those of Figure 5A and B neither affected the F3 band nor indicated the appearance of new slower migrating species, although the G-quadruplex arising from the other (+) strand (ODN4) was detected in K+ (lanes 6–9) and disappeared with DMS treatment (lane 12). Altogether these observations confirm that F'1 and F'2 correspond to flap structures containing G-quartets.



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  		Figure 5. Formation of G-quartets in flap substrates. Annealing and electrophoresis were carried out according to Materials and Methods. F indicates the position of the flap substrates, 1/2T are Watson–Crick duplexes. G'2 is an internal marker of G-quadruplexes adopted by ODN4 (30 µM) after 24 h incubation at 52°C in 200 mM KCl (lanes 1 in each panel). (A) F1 construction using mutODN(–) as template. Lanes 2 and 3, mutODN(–)/ODN3 and mutODN(–)/ODN4 duplexes; lane 4, F1 conserved at –20°C after annealing; lanes 5–10, F1 incubated for 35 h at 52°C in 50, 100, 200 or 400 mM KCl (lanes 6–10) or 100 or 400 mM LiCl (lanes 8–9); lanes 11 and 12, F1 incubated in 200 mM KCl containing, respectively, 0.005 and 0.02% DMS for 35 h at 52°C. (B) F2 construction using ODN(–) as the template for complete Watson–Crick annealing of the two (+) strands. Lanes 2 and 3, ODN(–)/ODN3 and ODN(–)/ODN4 duplexes. Other lane compositions were identical to the preceding experiment. (C) F3 construction using mutODN(–) and mutODN3. Lanes 2 and 3, mutODN(–)/mutODN3 and mutODN(–)/ODN4 duplexes. Other lane compositions were identical to the preceding experiment. (D) Fluorimaging quantification of F'1 and F'2 formation by increasing the amounts of KCl, as the ratio between F' species and the sum of F + F'.

 
F'1 and F'2 formation was efficiently promoted by a dibenzophenanthroline derivative (MMQ3), a selective G-quadruplex ligand
Incubation conditions (52°C for at least 24 h with up to 100 mM K+) in which the F' structures were previously obtained indicated that G-quartets were not easily processed. Therefore, we tried to induce their formation using a G-quadruplex interactive ligand of the dibenzophenanthroline family (MMQ3, see Fig. 6D). This dibenzophenanthroline series substituted by long polyammonium linkers has been described as being amongst the most efficient G-quadruplex stabilizing ligands (27). Therefore, F1 and F2 substrates were incubated at increasing concentrations of MMQ3 in 100 mM K+. As seen from Figure 6, addition of the G4 ligand induced a strong increase in the F'1 and F'2 species, up to 75% for F'1 and nearly 35% for F'2. This holds for a 2-fold less incubation time. This effect is in complete agreement with the G-quadruplex nature of the F' species.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study focused on the two adjacent repeats of guanines G6-G4 located in the sequence of the HIV-1 central DNA flap overlapping (+) strands, taking advantage of our reversible method to easily dissociate G-quartets with NaOH. As for other G-rich sequences, these G-tracts provoke K+ (or Na+)-dependent formation of G-quadruplexes in vitro. The G6 motif alone (ODN1) adopted a parallel interstrand conformation (G4) through the stacking of six G-quartets (Fig. 2), and ODN2, the G6-G4 repeats separated by 9 nt, can fold into either a parallel G4-DNA or a fold-back hairpin G'2 structure containing four G-quartets (Fig. 3). As predicted, ODN2 conversion from monomer to a distribution of G4 or G'2 quadruplexes was efficiently driven by the presence of K+ or Na+ but not by Li+ (14,15). Remarkably, even though the thermodynamic product of ODN2 conversion was a G'2-DNA, the parallel G4-DNA was the fastest to be formed in the presence of K+ or Na+ under our experimental conditions (Fig. 4). This suggests that G'2 formation passed through the intermediate of G4-DNA, as already proposed by Han et al. (29,30) in their experiments using the G-quartet ligand PIPER. Either the 9-nt spacer, the number of G residues or possible interstrand Watson–Crick base pairs may cap the dimer structure and add to the stability of the G'2-DNA described here. It is conceivable that this conformational shift could pass via the X species (Fig. 3), since its spontaneous conversion to either G4 or G'2 has been observed during its purification (not shown).

The HIV-1 central DNA flap is especially favorable for reverse branch migration after RT termination (22,28). This can drive a 3'-flap of the (+)U strand instead of a 5'-flap (Fig. 7A) and also put the G6-G4 clusters into close proximity. The K+ over-stabilization of G'2 structures for the G6-G4 sequences strongly suggested the possibility of transient G-quartets in the complete central DNA flap. Therefore, the F1 and F2 constructs formed shorts flaps focused on these G-tracts. Indeed, the overlapping (+) strands of these substrates were able to interact through G-quartets in the presence of K+, as evidenced by the formation of slower migrating species (Fig. 5A and B). This phenomenon did not occur in the presence of Li+, when the N7 of guanine was methylated or the G-tracts substituted in one strand, but was facilitated when the two (+) strands were not engaged in Watson–Crick base pairing with the template (50% F'1 versus 35% F'2; Fig. 5D). Therefore, G-quartet formation was enhanced to 75% for F1 and 40% for F2 in a significantly shorter time using the dibenzophenanthroline derivative MMQ3 (Fig. 6). Nevertheless, without MMQ3 the driving force of G-DNA structure formation in F2 must be branch migration, which imposes a structural fluctuation within the complementary strands and closely juxtaposes the two G6-G4 tracts enough to facilitate their association. Finally, we observed that concomitant annealing of the two overlapping strands was reduced for F2 (Fig. 5B) compared to F1 (Fig. 5A): this problem, which needs to be clarified, could be due to a destabilizing effect by the overlap on Watson–Crick bordering sequences.



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  		Figure 7. The reverse branch migration occurring in the HIV-1 central flap sequence can tie together the G6 and G4 tracts (black lines) of the two (+) strands. (A) Schematic illustration of the reverse branch migration in the flap substrates designed for this study. (B) Kissing hairpins and crossover-type G-quadruplex models proposed for G-quartet arrangements in flap substrates.

 
How can the HIV-1 genome take advantage of G-quadruplex structures within its central DNA flap? First of all, G-quadruplexes must be generated in the central DNA flap by strand displacement synthesis at the end of retrotranscription. The G-tracts are found in the INT coding sequence and are well conserved, especially the G6 one, among primate lentivirus genomes, according to a sequence alignment in the HIV sequence database (not shown). G-quartet formation does not require any extra stabilizing factors (acid pH, high salt, etc.), but was found in F1 and F2 substrates to be a slow process requiring thermal agitation. In vivo it could be monitored by selective proteins that stabilize G-quartets (7–9). Conversely, trapping of the (–) strand by cellular pyrimidine-rich ssDNA-binding factors (31) could favor, combined with branch migration, proximity of the two G6-G4 repeats. Figure 7B sketches our proposal of how G-quartets tie together the two (+) strands of the central DNA flap and give rise to two possible arrangements, a ‘crossover-type’ and a ‘kissing hairpins’ structure, according to the antiparallel association model of the strands in G'2-DNA. The ‘kissing hairpins’ structure seems quite attractive within this scheme, as the two 9-nt-long kissing loops are antiparallel and may form six potential Watson–Crick base pairs close to the G-quartets. Such a conformation could modulate the flap reactivity, by exposing either the G-quartets, the free ss(+) strand tail or the pyrimidine-rich (–) strand to cellular factors, for example nucleocytoplasmic shuttling proteins such as hnRNP K (pyrimidine strand) (32) and hnRNP A1 (G-quartets) (10), when such interactions should promote nuclear entry of HIV-1 PICs.

A G-quadruplex folding of the central DNA flap may resolve the question of its cytoplasmic resistance to cellular nucleases, considering that in vivo a complex machinery is devoted to the processing of flap structures (33). Flap endonuclease 1 (FEN-1) was shown to efficiently remove the 5'-tail of a HIV-1 central flap substrate in vitro when branch migration was blocked using non-homologous (+) strands (34). As was proposed for telomeres (1,3), a structure held by G-quartets may act as a capping site against nuclease degradation or may ‘freeze’ the branch migration toward a nuclease-resistant conformation. Effectively, G-quartets might allow neither FEN-1 activity (34,35) nor the RPA/DNA2 interaction needed for the cleavage of DNA flaps (33). Alternatively, G-quartets could be involved in the central flap as an enhancing substrate for selective uptake of HIV-1 PICs by the sub-nuclear NHEJ machinery (36). Interestingly, the NHEJ-associated Ku protein tightly binds and stabilizes G'2-quadruplexes (7), whereas it was found in purified PICs (36). Interactions of WRN helicase with Ku (37), as well as with FEN-1 (38) and with G-quadruplexes, could then drive dissociation of G-quartets, followed by processing of the 5' (+) strand flap (39). With its high potential affinity, quadruplex DNA should therefore constitute a novel virus control element inside the HIV-1 central DNA flap to efficiently promote the targeting of incoming HIV-1 DNA to nuclear machinery in charge of DNA maintenance. Apart from the HIV-1 DNA flap, our data also argue that G-quadruplexes may punctually form in genomic flap DNA that contains two adjacent runs of guanines, after strand displacement synthesis in Okazaki processing or in the long patch base excision repair pathway. In vivo experiments are obviously necessary to check these hypotheses.


   ACKNOWLEDGEMENTS
 
We thank Etienne Delain (I.G.R., Villejuif) and Laurent Lacroix (M.H.N, Paris) for helpful comments on the manuscript. The work was supported by grants from the Agence Nationale de Recherche sur le SIDA (ANRS). S.L. is the recipient of a fellowship from the ANRS. G.M. is Assistant Professor at the Université Pierre et Marie Curie (Paris).


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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