Nodule DNA in the (GA)37*(CT)37 Insert in Superhelical Plasmids*

Di- or trivalent metal ions stabilize a supercoil-de-pendent transition in pGA37, which contains the (GA)s7*(CT)s7 insert, at neutral and basic pH. The structure formed is different from the well known protonated triplexes (H-DNA) adopted at low pH by polypurine-polypyrimidine (Pur-Pyr) inserts in plas- mids. DNA samples must be preincubated in the presence of multivalent ions at 60 OC for the new transition to occur. At neutral pH in the presence of Co hexamine, both strands of the insert have modification maxima situated at one-third of the distance from both ends. We propose the formation of a new structure called nodule DNA which consists of both Pyr*Pur*Pyr and Pur*Pur*Pyr triplexes and does not contain continuous single-stranded regions. At basic pH (>8.6) in the presence of magnesium ions, the modification pattern corresponds to Pur*Pur-Pyr triplex formation in the whole insert. At neutral pH in the presence of magnesium, both nodule DNA and the Pur-Pur-Pyr triplex can be formed in the insert. We also observed a magne-sium-dependent transition at neutral pH in the other Pur-Pyr insert containing plasmids. These data demonstrate that Pur-Pyr sequences can adopt several non-B conformations at close to in vivo conditions.

pUC9 vector forms H-DNA at native superhelical density (-0.05 to -0.06) and at pH values lower than 6.5 (16). On the other hand, these sequences were also hypersensitive to nucleases even at basic pH (13).
Herein, we describe the structural transitions of the (GA)37. (CT)37 insert at neutral and basic pH values, utilizing chemical probe analyses which provide resolution to the base pair level. A novel DNA conformation (nodule DNA) was identified.

MATERIALS AND METHODS
PZnsmids"pGA37 (16) and pGA2O (13) containing (GA)37.(CT)3, and (GA),o. (CT),, inserts in the EcoRI site of pUC9 were described. pRW1716 and pRW1724 were constructed in this laboratory (17). All plasmids were isolated from Escherichia coli HBlOl as described (17). Samples with different degrees of negative supercoiling were obtained using relaxation by topoisomerase in the presence of different concentrations of ethidium bromide (18). The average superhelical density was estimated by agarose gel electrophoresis in the presence of chloroquine phosphate (19).
Chemical Modifications-For the standard experiments, a sample contained 1 pg of the plasmid in 40 mM Tris.HC1 buffer (pH 7.5 or 9.0), 20 mM NaCl plus one of the following: 10 mM Mg&, 10 mM CaC12, 1 mM c o hexamine (Sigma), or 1 mM spermidine (Sigma).
Modifications by oso4, DEPC, and KMn04 were performed as described (20). Briefly, samples were modified in the total volume of 50 pl with 2.5 mM 0 8 0 4 plus 2.5 mM 2,2'-dipyridyl for 5 min, 2 mM KMn04 for 2 min, or 1 pl of DEPC for 5 min, all at room temperature. 0 8 0 4 reactions were stopped by ether extraction. DEPC and KMn04 reactions were stopped by addition of 8-mercaptoethanol to 1 M and ethanol precipitation. All samples were purified by gel filtration through Sephadex G-50 microcolumns equilibrated with H20 (21). Digestion by PI nuclease (BRL) was performed in a buffer containing 50 mM Tris.HC1 (pH 7.5), 20 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, and 10 mM MgCl, with 15 units of PI nuclease in a total volume of 50 pl. Reactions were stopped by extraction with phenol chloroform and DNA was purified by gel filtration through G-50 microcolumns.
In the majority of the experiments for the analysis of the modification in the Pur.Pyr inserts, modified plasmids were cut by SalI, labeled at the 3'-end (for Pyr-strand analysis) by the Klenow fragment of DNA polymerase I (U. S. Biochemicals) or at the 5'-end (for Pur-strand analysis) by T4 polynucleotide kinase (U. S. Biochemicals), and cut by HindIII. After purification on G-50 microcolumns, heating in piperidine and lyophilizations, samples were analyzed on sequencing gels without fragment purification, allowing the shorter (15 base pairs) fragment to run off the gel. In other cases, the shorter HindIII-EaeI fragment of the modified pGA37 was purified from polyacrylamide with subsequent analyses on sequencing gels. The autoradiograms of the gels were quantitated as described (22).

RESULTS
Transition in (GA)37. (CT)37 Insert after Preincubation in hfagnesium"OsO4, KMn04, DEPC, and P1 nuclease were used as probes for the structures of the Pur. Pyr inserts since they are sensitive reagents for bases in unusual conformations (20,24,26). os04 and KMn04 were used as probes for the pyrimidine strand of the inserts since they react predomi-nantly with thymines, whereas DEPC was used as a probe for the purine strand since it reacts with adenines and guanines in non-B conformations. Fig. 1 shows modification of pGA37 at native superhelical density (-0.055) by different probes. For the samples marked -, MgC12 was added at room temperature just prior to modification. Samples marked + were preincubated in the presence of MgC12 at 50 "C, cooled to room temperature, and then modified. Only background levels of modification were found for the samples that were not preincubated (lanes 1,4,6, and 8). However, after preincubation in the presence of magnesium, a substantial increase in reactivity was observed with all probes in the central part of the insert. 0 s 0 4 modification (lane 2) reveals three maxima; one is situated at the center of the insert and two others are at one-third of the way from the ends. However, after additional preincubation in the presence of EDTA, the modification returns to the background level (lane 3).
KMn04 and DEPC modification patterns (lanes 5 and 9) are similar to those found with oso4, except that the onethird maximum closer to the 3'-end (for GA-strand) is weaker than the one closer to the 5'-end. In the case of PI nuclease,  and 7), and DEPC (lanes 8 and 9). All reactions were performed at room temperature in buffers containing 10 mM MgC12 (see "Materials and Methods"). The samples in lanes 2,5, 7, and 9 were preincubated at 50 'C for 30 min before modification. The sample in lane 3 was preincubated in the presence of MgC12 followed by incubation at 50 "C for 30 min in the presence of 20 mM EDTA. Samples were analyzed on sequencing gels without fragment purification (see "Materials and Methods"). Thus, preincubation of pGA37 in the presence of magnesium results in a dramatic increase in the reactivity of certain bases, reflecting a change in the structure of the (GA)37-( CT)37 insert.
Kinetics of the Structural Changes- Fig. 2 presents the time course of the oso4 modification of the plasmid preincubated in a buffer containing 10 mM MgCI2 at 50 "C. Only a background level of reactivity of the CT-strand of the insert was found without preincubation. However, after as little as 2 min of preincubation, an increase in the reactivity of thymines inside the central one-third of the insert is observed but there is no distinct maxima or minima. After 5 min of preincubation, three maxima of modification inside the insert are found. These maxima get sharper with an increase in the preincubation time. No differences were observed between the modification patterns after 30 and 80 min of preincubation. After 80 min preincubation at 37 "C, the modification pattern resembles that found after 2 min preincubation at 50 "C with no distinct maxima (data not shown). Effect of Different Ions- Fig. 3 (left panel) shows the os04 and DEPC modification patterns of pGA37 at native superhelical density in the presence of 1 mM Co hexamine. In both cases, the EaeI-Hind111 fragment containing the insert was labeled on the purine (3'-EaeI) or pyrimidine (3"HindIII) strand, purified, and analyzed on a sequencing gel. Only background levels of modification were observed after the reactions without preincubation (lanes 2 and 4 ) . After preincubation in the presence of Co hexamine, both strands have  maxima of modification situated one-third of the way from the ends. two-dimensional gel electrophoresis of topoisomers in the presence of magnesium (data not shown). No transition was observed in topoisomers up to number -15 which corresponds to a superhelical density of -0.056. However, more information could not be derived from the two-dimensional gels because of the low resolution of the highly negative supercoiled topoisomers. Thus, we took advantage of the relatively long kinetics of pGA37 in order to determine the amount of relaxation caused by the transition. distributions and therefore the amount of relaxation introduced by the transition corresponds to 6.5 topoisomers for both magnesium and Co hexamine containing samples.
Transition at Basic pH- Fig. 6 shows the modification of pGA37 by oso4 and DEPC at pH 9.0. For the analysis of the purine strand, the Ed-HindIII fragment labeled at the 3'-EaeI end was purified. The pyrimidine strand was analyzed without fragment purification (see "Materials and Methods"). If the samples were not preincubated, then only a background level of modification was observed. Reaction of preincubated DNA with DEPC revealed only one maximum of modification which was at the center of the purine strand, whereas the rest of the insert was protected from modification even in comparison with the unpreincubated sample. os04 modification revealed the maximum at the center of the pyrimidine strand and stronger reactivity of thymines with the 5'-half compared with 3'-half of the insert. Experiments with Other Plasmids-0sO4 modification experiments were also carried out with pGA20, pRW1724, and pRW1716. pGA2O is the same as pGA37, except that it contains (GA)20 insert instead of (GA)37. pRW1724 and pRW1716 were obtained by introducing (AGG),TCC(AGG)s and (GAA),TTCGC(GAA), inserts into the single BamHI site of the pRW791 vector (20). Thus, inserts were studied with approximately 33, 50, and 66% G + C. Fig. 7 presents os04 modification patterns of pGA20, pRW1724, and pRW1716. Preincubation of pGA2O at 50 "C in the presence of 10 mM MgC12 results in maximum modification at the center of the (GA)20. (CT)20 insert and some protection of the thymines in the rest of the insert. The intensity of the modification maximum in the case of pGA20 was considerably less than found with pGA37.
In the case of pRW1724 and pRW1716, the modification of the (AGG),TCC(AGG)s and (GAA)gTTCGC(GAA)S inserts, respectively, was analyzed. Preincubation of pRW1724 in the presence of 10 mM MgC12 resulted in hypermodification of thymine in the TCC sequence situated between the two runs of purines. No increase in reactivity of thymines between the two runs of purines was observed for pRW1716. Also, the background modification of purines decreased after preincubation in the presence of magnesium. Therefore, preincubation in the presence of magnesium caused structural changes not only in (GA)37 but also in the other Pur. Pyr inserts. For these changes to occur, a Pur Pyr insert must be GC-rich. Also the distribution of Os04-hypersensitive sites is different for (GA)37 and the shorter inserts, which reflects the difference in the nature of structural changes (see "Discussion").

DISCUSSION
These data demonstrate that supercoil-dependent structural transitions occur in a (GA)37.(CT)37 insert at neutral and basic pH in the presence of dior trivalent ions. These transitions are quite slow, requiring preincubation of the plasmids at 50 "C for at least 20 min. Preincubation of pGA37 in the presence of MgC12 at pH 9.0 results in a DEPC modification maximum at the center of the purine strand and an increase of reactivity with Os04 at the 5'-half of the pyrimidine strand with the maximum reactivity at the center of the insert. This modification pattern corresponds to the formation of an intramolecular Pur e Pur. Pyr triplex between the 3'-half of the insert and the 5'-half of the purine strand with the 5'-half of the pyrimidine strand remaining singlestranded. The 5'-half of the purine strand (the third strand of the triplex) binds to the 3'-half of the insert via Hoogsteenlike hydrogen bonds, forming G G . C and A. A. T base triads  1 1 1 1 1 1 1 1 1 1 1 1 1 1   1  (27). The formation of co-linear intermolecular Pur. Pur. Pyr triplexes stabilized by these triads was described (28,29). In the case of an intramolecular triplex, the two purine strands are antiparallel and therefore part of the purines in the third strand must be in the syn conformation (30). This type of triplex was observed previously for a G,.C, insert in the presence of magnesium at neutral pH (8, 12). Preincubation of pGA37 at pH 7.5 in the presence of Co hexamine results in two maxima of reactivity with both Os04 and DEPC (for the pyrimidine and the purine strands, respectively) situated one-third of the way from both ends of the insert. To explain these results, we propose a novel bitriplex structure formed by strand exchange, called nodule DNA (Fig. 8). This structure is composed of both a protonated Pyre Pur -Pyr triplex and a Pur -Pur Pyr triplex. Nodule DNA does not contain regions of single-stranded DNA except in the loop regions located at a distance of one-third from the ends of the insert. These loops are hypersensitive to the chemical and enzymatic probes. The formation of nodule DNA reduces the distance between the 5'-and 3'-ends, therefore causing a shortening of DNA molecule. Previous studies (17) have characterized more simple types of bitriplexes which do not require strand exchange.
The formation of nodule DNA and the Pur Pur Pyr triplex is in agreement with the amount of relaxation caused by the transition of the insert as determined by agarose gel relaxation studies. Both nodule DNA and a Pur -Pur. Pyr triplex are topologically equivalent to the melted state of the insert.
Melting of the 74-base pairs insert corresponds to the relaxation of 7.0 turns, assuming 10.5 base pairs per turn in the duplex state. This value is close to the 6.5 turns of relaxation which were experimentally observed (Fig. 5). The discrepancy of 0.5 turns may be caused by distortions at the ends and/or by the difference from 10.5 for the number of base pairs per turn for the duplex state of the insert. In any case, the amount of relaxation observed reveals that the transition involves the entire insert. Therefore, possible models that consider triplex formation in two-thirds of the (GA)37 insert can be ruled out. The modification pattern observed at neutral pH in the presence of MgClz corresponds to a mixture of the Pur -Pur.
Pyr triplex and nodule DNA. Fig. 9 illustrates the different structures than can be formed in the (GA)37. (CT)37 insert. At a pH lower than 6.5 (16), the Pyr-Pur. Pyr triplex which requires protonation of cytosines was formed. At basic pH, the Pur-Pur .Pyr triplex was formed which required magnesium or other multivalent ions for stability. At neutral pH, nodule DNA, containing both types of triplexes, can be formed. All these structures are separated by high energetic barriers. Therefore, once one of these conformations is adopted by a given sequence, it is kinetically trapped, and therefore two or even three structures may coexist in the same plasmid population. Numerous examples of the occurrence of long (GA), sequences in regulatory regions of eukaryotic genomes are known (1,31,32). The structural polymorphism (four conformations, Fig. 9) of these sequences may be involved in genetic regulation. Different proteins may selectively bind to the different conformations of (GA),. Alternatively, the transition to the non-B conformations may cause a displacement of proteins (ix. histones) that normally bind to DNA.