Plasmid DNA

A region containg 522 bp Pyr-Pur stretch (−2, 201 to −1 , 680) of the Ars gene of the sea urchin (Hemicentrotus puicherrimus) was excised and inserted into pBluescript SK–(pBSK) vector (Stratagene Cloning Systems, USA) to construct the plasmid pBSK-AvNs (Fig. 1). Various degrees of negative supercoiling were introduced into pBSK-AvNs by treating 20 μg/ml of pBSK-AvNs with topoisomerase I at 37°C for 1 hr in the presence of various concentrations of ethidium bromide. After extracting the supercoiled DNA by phenol/chloroform, ethidium bromide was removed by water-saturated n-butanol, and DNA was precipitated and washed with ethanol, and dried in vacuo.

Fig. 1.

Restriction sites around the Pyr-Pur region in the Ars gene and in the pBSK-AvNs. Slashed box; the Pyr-Pur region, open boxes; exons, a thick line; 5′ flanking region and introns, a thin line; pBSK vector. A; Apal, H; HindIII, N; Nsp75241, Av; Aval, E; EcoRI.

The density of negative supercoiling was determined by two dimensional electrophoresis (Pulleyblank et al., 1985). A mixture of variously supercoiled plasmids was first electrophoresed on 1.5% agarose in 0.5 × TBE, then gels were equilibrated with 1 μg/ml chloroquine in 0.5 × TBE. The second electrophoresis was performed in the presence of 1 μg/ml chloroquine in 0.5 × TBE. The DNA bands were detected by southern blotting with digoxygeninlabeled pBSK-AvNs as the probe.

Diethylpyrocarbonate (DEPC) and osmium tetroxide reactions

Modification of purine bases (especially adenine) by DEPC and pyrimidine bases (especially thymine) by osmium tetroxide in the negatively supercoiled pBSK-AvNs were carried out as described by Hanvey et at. (1988) with some modifications.

Three μg of negatively supercoiled pBSK-AvNs was dissolved in 100 μl of 0.3 M Tris-HCI buffer (pH 7.5) containing 5 mM MgCl₂ and 0.25 M KCl, and incubated for 1 hr at 13°C. The DEPC reaction was started by adding 10 μl of DEPC to the mixture, followed by 1min of vortexing and a 30-min incubation at 22°C. The osmium tetroxide reaction was started by addition of 2 μl of pyridine and 10 μl of 20 mM OsO₄ to the mixture, followed by 1-min vortexing and 30-min incubation at 22°C. The plasmid DNA was then cleaved at modified nucleotides by piperidine, and sites of modification were determined on a denaturing polyacrylamide gel.

Electrophoretic mobility of negatively supercoiled pBSK-AvNs

pBSK-AvNs was negatively supercoiled by DNA topoisomerase I treatment in the presence of ethidium bromide and electrophoresed on 1.5% agarose gel containing TBE buffer at pH 8.2. Increasing the concentration of ethidium bromide, increased the electrophoretic mobility of pBSK-AvNs (Fig. 2), indicating that the level of negative supercoiling of pBSK-AvNs is positively dependent on the concentration of ethidium bromide in the topoisomerase reaction. Electrophoretic mobility of the plasmid decreased by increasing ethidium bromide from 2 μg/ml to 10 μg/ml, suggesting that the high negative supercoiling (σ<−0.0385, as determined in the presence of chloroquine by two dimensional electrophoresis, see Materials and Methods) causes a severe conformational change of the plasmid that leads to reduction of the electrophoretic mobility. A plasmid with this level of negative supercoiling is hereafter referred to as highly negatively supercoiled, with the supercoiling density of σ=−0.0357 as a native level of supercoiling.

Fig. 2.

Electrophoretic mobility of pBSK-AvNs changes depending on degree of negative supercoiling. Various degrees of negative supercoiling were produced in pBSK-AvNs by treating the plasmid with topoisomerase I in the presence of various concentrations of ethidium bromide. Electrophoresis was carried out in 1.5% agarose gel. Concentration of ethidium bromide, lane 2; without ethidium bromide, lane 3; 0.2 μg/mg, lane 4; 0.4 μg/ml, lane 5; 0.6 μg/ml, lane 6; 0.8 μg/ml, lane 7; 1.0 μg/ml, lane 8; 1.2 μg/ml, lane 9; 1.4 μg/ml, lane 10; 2 μg/ml, lane 11; 5 μg/ml, lane 12; 8 μg/ml, lane 13; 10 μg/ml. Lanes 1 and 14; pBSK-AvNs before treatment with topoisomerase I.

Chemical modification of bases in negatively supercoiled pBSK-AvNs

It is known that diethylpyrocarbonate (DEPC) carboxyethylates adenines at their N7 and cleaves an imidazole ring, while OsO₄ reacts with thymines at their C5 and C6 to form osmate esters, when DNA is in a non-B structure (Johnston, 1988; Vojtiskova and Palecek, 1987; Wells et al., 1988). Thus the sensitivity of DNA to these reagents is a good measure to estimate its structure.

Modification by DEPC and OsO₄ of nucleotide bases in a linearized pBSK-AvNs, a native pBSK-AvNs (with a native level of supercoiling) and a highly negatively supercoiled pBSK-AvNs was surveyed under physiological ionic strength and Mg²⁺ concentration similar to in vivo levels. The experimental results are shown in Figure 3, and the positions of modified bases are summarized schematically in Figure 4.

Fig. 3.

Base modificaiton of Pyr-Pur region in negatively supercoiled plasmid by DEPC or by OsO₄. pBSK-AvNs in linear, native and highlynegatively supercoiled forms were treated with DEPC or with OsO₄, cleaved by HindIII, labeled with ³²P at the 3′ end of the pyrimidine strand, and the labeled HindIII-Apal fragments were isolated (a and b). In the case of preparation of the ³²P-labeled HindIII-Apal fragments from the purine strand (c and d), the plasmid was first cut by Apal, and the labeled fragment was prepared in a similar way. The labeled fragments were cleaved by piperidine at modified bases and electrophoresed on 4% denaturing polyacrylamide gel (a and b) and on 6% denaturing polyacrylamide gel (c and d). Sites of (CT)₁₁ or (GA)₁₁ were indicated by vertical lines on the right side of each column. Lanes 1 and 2; Maxam-Gilbert ladders or A+G and C in (a) and (b) and C+T and G in (c) and (d), respectively. Lane 3; linearized pBSK-AvNs, lane 4; native pBSK-AvNs, lane 5; highly negatively supercoiled pBSK-AvNs. (a) DEPC-sensitive sites in the pyrimidine strand. (b) OsO₄-sensitive sites in the pyrimidine strand. (c) DEPC-sensitive sites in the purine strand. (d) OsO₄-sensitive sites in the purine strand.

Fig. 4.

Mapping of sites of base modification in HindIII-Apal fragments in linear, native and highly negatively supercoiled plasmids. The sites of base modification determined in experiments in Fig. 3 are mapped in the nucleotide sequence of AvNs fragments. Sites of base modification in (a) a linearized plasmid, (b) a native plasmid, and (c) a highly negatively supercoiled plasmid. Thin and short arrows indicate weakly sensitive sites, and thick and long arrows indicate highly sensitive sites. Very thick horizontal arrows indicate a mirror repeat sequence.

Clustered adenines in the purine strand of a linear DNA were chemically modified while any bases in the pyrimidine strand were not modified (Fig. 4a). At present we do not have any evidence to explain increased accessibility of DEPC to adenines in linear DNA fragments, but it is conceivable that consecutive adenines tend to cause DNA bending (Sinden, 1994) that would make adenine bases accessible to DEPC.

In the case of a native pBSK-AvNs (Fig. 4b), though the purine strand was modified by DEPC in a manner similar to a linear plasmid, thymines in the pyrimidine strand that were not accessible to OsO₄ in a linear plasmid became accessible to the reagent. It is of interest that, while the (CT)₁₁ tract was extensively modified by OsO₄, the complementary (GA)₁₁ tract was not modified by DEPC, and thymine stretches flanking both ends of the (CT)₁₁ tract were protected from modification. This result suggests that even a native level of negative supercoiling can produce a conformational change in the Pyr-Pur region which results in a DNA structure slightly deviated from B-form.

By imposing higher negative supercoiling than native level, the accessibility of base-modifying reagents to the plasmid increased in the purine strand. As depicted in Figure 4c, while little change was observed in the thymine modification of the pyrimidine strand as compared with a plasmid with a native level of supercoiling, a marked change in adenine modification was observed in a restricted region of the purine strand. Adenines in the 3′ half of (GA)₁₁ in the purine strand were modified and the adenines upstream of (GA)_n (from −1,827 to −1,807) became more accessible to DEPC. That is, adenines between −1,827 and −1,793 were all modified, indicating the single-stranded nature of the purine strand of this region under high negative supercoiling.

A triplex DNA structure of the Pyr-Pur region

A triplex DNA structure through intramolecular formation of Hoogsteen base-pairings (Hoogsteen, 1963) seems to be a most plausible DNA model that accomodates most of the above experimental results on the accessibility of the Pyr-Pur region to base-modifying reagents. A possible model of a triplex DNA structure taken by the Pyr-Pur region of the sea urchin Ars gene is depicted in Figure 5. In this model, the purine strand between −1,829 and −1,787 is single-stranded, while the pyrimidine counterpart winds up around the upstream double helical DNA along its major groove to form a triple-stranded DNA structure. Thymines in the third pyrimidine strand of this structure would be eventually protected from attack by OsO₄.

Fig. 5.

A possible H-DNA model formed in polypyrimidine region of the sea urchin Ars gene. Small arrows indicate bases weakly accessible to base-modifying reagents when the polypyrimidine region is highly negatively supercoiled, and large arrows indicate bases highly accessible to the reagents.

The existene of a mirror repeat sequence (from −1,849 to −1,804; Fig. 4c) in the Pyr-Pur region supports this model. If strand separation occurs in the right half of the mirror repeat accompanied by folding back of its single-stranded pyrimidine counterpart along a major groove of the left half of the mirror repeat, 21 Hoogsteen base-pairings containing C-G-C and T-A-T would be formed between the purine strand of the double helix and the pyrimidine strand that folded back over it, as suggested by Wells et al. (1988). In spite of the presence of Mg²⁺, H-DNA structure appears to be formed through C-G-C and T-A-T in the Pyr-Pur region of the sea urchin Ars gene, while Kohwi and Kohwi-Shigematsu (1988) showed that in the presence of Mg²⁺ an isomer of HONA is formed that contains C-G-G in place of C-G-C. Alternately-arranged T-A-T's may stabilize the C-G-C structure.

Biological significance of a triplex DNA structure is not clear though its possible functions in transcription, replication and recombination have been discussed (Sinden, 1994). Michel et al. (1993) suggested that a triplex DNA region may act as a transcriptional insulator by insulating some genetic loci from influences of other genes through its unusual steric conformation, while Baran et al. (1991) showed that DNA synthesis is arrested at sites of DNA triplex formation. It is also possible that sharp bends of DNA in these structures may help interaction of protein factors that bind to distantly separated cis-elements.