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NONSTANDARD DNA

STRUCTURES A N D T H E I R ANALYSIS

[8]

Detection o f Z-DNA by Restriction Methylases

Restriction methylases are unable to methylate their target sequence if it is in the Z conformation.78 This observation has been used to assay for Z-DNA formation in supercoiled plasmids in vivo. Jaworski et aL 79 and Rahmouni and Wells72 introduced a plasmid expressing a temperaturesensitive EcoRI methylase into E. coli and showed that, when grown at the permissive temperature, all available EcoRI sites were methylated. When cotransfected with plasmids containing alternating CG sequences of different lengths adjacent to an EcoRI site, those plasmids having large CG inserts were not methylated in vivo (as determined by susceptibility to EcoRI endonuclease after isolation of the plasmid) at the permissive temperature, indicating that they existed in the Z form in vivo. Acknowledgments I thank A. Rich for suggestions,and A. Rich and G. J. Quigleyfor critical readingof the manuscript. This work was supported in part by National Institutes of Health Grant 1R29GM41423. 7sL. Vardimonand A. Rich, Proc. Natl. Acad. Sci. U.S.A.81, 3268 (1984). 79A. Jaworski, W.-T. Hsieh, J. A. Blaho,J. E. l_arson, and R. D. Wells, Science 238, 773 (1987).

[8] S u p e r c o i l e d

DNA and Cruciform Structures

By ALASTAIRI. H. MURCHIE and DAVID M. J. LILLEY

Supercoiled DNA and Local DNA Structure The sequence-dependent rearrangement of DNA structure to adopt a new geometry usually involves a change in local DNA twist, and this is almost always negative; that is to say, perturbed DNA structures are usually underwound relative to the normal B-form double helix. For this reason, such structures will be more stable in negatively supercoiled DNA circles than their relaxed counterparts, and DNA supercoiling is well known to stabilize a number of structural polymorphs, including lefthanded Z-DNA, ~,2 cruciform structures, 3-5 and H-triplex structures. 6 The I L. J. Peck, A. Nordheim,A. Rich, and J. C. Wan~ Proc. NatL AcaR ScL U.S.A. 79, 4560 (1982). 2C.K. Singleton,J. Klysik, S.M. Stirdivant, and R. D. Wens, Nature(London) 299, 312 (1982). METHODS IN ENZYMOLOGY, VOL. 211

Copydght © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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basis of this stabilization lies in simple topology. A circular doublestranded molecule can exist in a number of isomeric forms (topoisomers) that differ in the number times one strand is linked with the other (linking number, L k ) . For an unconstrained, relaxed DNA molecule, the linking number is given by Lk = Lk ° = N/h

(1)

where N is the number of base pairs in the circle, and h is the helical repeat under the conditions of the experiment. Note that although L k is required to be an integer, N / h may not be so, and thus a completely relaxed topoisomer may not be attainable for a particular sized molecule under a certain set of conditions of temperature, ionic strength, etc. It is useful to define the linkage of the molecule relative to the relaxed state, by means of the linking difference (ALk): ALk

= L k -- L k °

(2)

For the reasons above, the linking difference need not necessarily be an integer. The linking difference measures the number of helical turns by which a given topoisomer differs from the relaxed state. Most natural DNA molecules are underwound, or negatively supercoiled, so that A L k has negative sign. To provide a measure of linkage changes that is independent of molecular size, we define the specific linking difference or superhelix density (tr): a = ALk/Lk

(3)

°

Many natural DNA molecules extracted from cells have a level of supercoiling corresponding to an underwinding of about 1 turn in 20, that is, a superhelix density o f - 0 . 0 5 . Energy is required to supercoil DNA. The free energy of a supercoiled DNA molecule (AGO relative to the relaxed state is quadratically related to the linking difference,7-9 namely, AG, = ( I050RT/N)

ALk 2

(4)

3 M. Gellert, K. Mizuuchi, M. H. O'Dea, H. Ohmori, and J. Tomizawa, Cold Spring Harbor Syrup. Quant. Biol. 43, 35 (1979). 4 D. M. J. Lilley, Proc. Natl. Acad. Sci. U.S.A. 77, 6468 (1980). 5 N. Panayotatos and R. D. Wells, Nature (London) 289, 466 (1981). 6 V. Lyamichev, S. M. Mirkin, and M. D. Frank-Kamenetskii, J. Biomol. Struct. Dyn. 3, 667 (1986). R. E. Depew and J. C. Wang, Proc. Natl. Acad. Sci. U.S.A. 72, 4275 (1975). s D. S. Horowitz and J. C. Wang, J. Mol. Biol. 173, 75 (1984). 9 D. E. PuUeyblank, M. Shure, D. Tang, J. Vinograd, and H.-P. Vosberg, Proc. Natl. Acad. Sci. U.S.A. 72, 4280 (1975).

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NONSTANDARD D N A STRUCTURES AND THEIR ANALYSIS

[8]

where R is the gas constant and T the absolute temperature. The alteration in linkage requires geometric changes to occur in the molecule, of torsional or flexural character, and this is stated mathematically by A L k = A T w + Wr

(5)

where T w is the helical twist and Wr is the writhing of the helix axis in three dimensions. Although this treatment has recently been absorbed into a much more detailed analysis of the topology of circular DNA, 1° it is adequate to understand the basis of the stabilization of certain perturbed structures, such a cruciforms, in supercoiled DNA. Because such structures are associated with a local unwinding, the negative twist change helps to compensate for the linkage deficiency by Eq. (5) and thus the molecule becomes partially relaxed by virtue of t h e change in topology. Thus the energetic cost of forming the new DNA structure locally (assumed to be positive) may be balanced by the negative free energy change associated with the global relaxation of the molecule, which can be calculated from Eq. (4). Because the supercoiling energy that is available for a given amount of relaxation increases quadratically with linking difference, the structure will be stable at a level of supercoiling that is greater than some critical value. Thus it is generally observed that structures such as cruciforms, which are unstable in relaxed or linear DNA, are formed cooperatively at a threshold level of supercoiling.

Cruciform Structures: Energetics and Kinetics Cruciform structures are paired hairpin loop structures, formed by intrastrand base pairing that is possible when a sequence possesses 2-fold symmetry. An example is shown in Fig. 1. When an inverted repeat containing a total of n bases (stem plus loop) forms a cruciform structure, there is a local unwinding of the DNA (ATwc) that is approximately given by ATwc = n/h

(6)

The free energy due to this relaxation for a given topoisomer can be calculated from Eq. (4), and this is offset against the free energy of cruciform formation (AG,), which will be the cost of forming two hairpin loops and a four-way junction. For inverted repeats of average sequence and base ~oj. H. White, N. R. Cozzarelli, and W. R. Bauer, Science 241, 323 (1988).

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SUPERCOILED D N A AND CRUCIFORM STRUCTURES

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S' A A A G T f X ~ T ~ T C C A A A T ~ T T G ~ T ~ C A A 3' T T T ~ T C G T T A C - K ~ T T T A C C C T ~ T C C T C ~ ; T T

S

A A A

AAA ~T

T G CG CG TA AT AT CG GC AT TA CG CG TA GC

CAA G~

CG AT GC GC AT TA CG GC TA TA AT GC GC T T

C A T

FIG. 1. Formation of a cruciform structure by an inverted repeat. The base sequence of the ColEI inverted repeat is shown along with its cruciform conformation? ,5

composition AGe has been measured to be 17-18 kcal mol-~, l~-~a although this should be regarded as a lower limit, since the introduction of longer loops or other imperfections into the 2-fold symmetry mean that there is no real upper limit. The energetic limits mean that at normal levels of superhelix densities ( - a - 0.06) a cruciform will not usually be detected " A. J. Courey and J. C. Wang, Cell (Cambridge, Mass.) 33, 817 (1983). ~2 M. Gellert, M. H. O'Dea, and IC Mizuuchi, Proc. Natl. Acad. Sci. U.S.A. 80, 5545 (1983). ~3D. M. J. Lilley and L. R. Hallam, J. Mol. Biol. 180, 179 (1984).

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NONSTANDARD D N A STRUCTURES AND THEIR ANALYSIS

[8]

if it has a stem shorter than 10- 11 base pairs (bp), a loop longer than 5 - 6 bases, and greater than 5% mismatching in the stems. For special sequences AGe can be lower; AGe was measured to be around 13.5 kcal mol -~ for alternating adenine-thymine sequences, ~4,~5 whereas for a (GATC)~0 sequence a value of 16 kcal mol -I was found. 16 Significant kinetic barriers to cruciform extrusion exists, t1'12'17-19 and the extrusion can be a very slow process. We found that the kinetics were markedly dependent on local DNA sequence and base composition, and we observed that the extrusion of the majority of sequences could be divided into two kinetic classes, 19 namely, S-type and C-type extrusion. The great majority of sequences are S type. These sequences do not undergo extrusion in the absence of salt, requiring 50-60 m M sodium for optimal extrusion rates. Under these conditions they exhibit moderate temperature dependence, with activation energies in the 30- 60 kcal molrange. An alternative kinetic behavior was found to be associated with inverted repeats that were flanked by (A + T)-rich DNA sequences.2° These sequences undergo extrusion with maximal rates at low ionic strength and are suppressed by salt. C-Type extrusion has a very high temperature dependence, with activation energies in the 100-200 kcal mol -~ range. We proposed two mechanistic schemes to account for these findings (Fig. 2). These are reviewed in detail by Lilley.2~ The S-type sequences are proposed to undergo a local opening at the center of the inverted repeat, followed by the initial formation of a junction structure to form the protocruciform. This may then extrude to the fully formed cruciform structure by a branch migration process. The smaller degree of initial opening is consistent with the relatively low activation energy and with the unwinding estimated by Courey and Wang22; it is also consistent with observations that sequence changes22-24 and base modifications25 in the center of the inverted repeat have the greatest kinetic consequences. We demonstrated that the rate of S-type extrusion is proportional to the anhy14 D. R. Greaves, R. K. Patient, and D. M. J. Lilley, J. Mol. Biol. 185, 461 (1985). 15j. A. McClellan, E. Palecek, and D. M. J. Lilley, Nucleic Acids Res. 14, 9291 (1986). 16 L. H. Naylor, D. M. J. Lilley, and H. van de Sande, EMBO J. 5, 2407 (1986). 17 R. R. Sinden and D. E. Pettijohn, J. Biol. Chem. 259, 6593 (1984). is I. Panyutin, V. Klishko, and V. Lyamichev, J. Biomol. Struct. Dyn. 1, 1311 (1984). 19 D. M. J. Lilley, Nucleic Acids Res. 13, 1443 (1985). 2o K. M. Sullivan and D. M. J. LiUey, Cell (Cambridge, Mass.) 47, 817 (1986). 21 D. M. J. Lilley, Chem. Soc. Rev. 18, 53 (1989). 22 A. J. Courey and J. C. Wang, J. Mol. Biol. 202, 35 (1988). 23 A. I. H. Murchie and D. M. J. LiUey, Nucleic Acids Res. 15, 9641 (1987). 24 G. Zheng and R. R. Sinden, J. Biol. Chem. 263, 5356 (1988). 25 A. I. H. Murchie and D. M. J. Lilley, J. Mol. Biol. 205, 593 (1989).

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SUPERCOILED DNA

ANDCRUCIFORMSTRUCTURES

163

C-type extrusion closure traps

large-scale opening A+T rich D N /

I

invertedrepeat,B-form

~_~cruciform

central ~ base opening

branch

9

migration

hairpin formation S-type extrusion

FIG. 2. Mechanistic schemes for cruciform extrusion. Kinetic data indicate that cruciform extrusion may proceed via two alternative mechanismsJ 9.2° The majority of sequences undergo cruciform extrusion by the S-type pathway in which there is a relatively contained initial opening (probably around 10 bp) at the center of the inverted repeat, followed by intrastrand base pairing and junction formation. The structure may then undergo branch migration to generate the fully extruded cruciform. This pathway requires salt. A small number of sequences, in which the inverted repeat is contiguous with (A + T)-rich DNA, may extrude via the C-type mechanism. At low salt concentrations the (A + T)-rich DNA may initiate thermal helix opening of a relatively large region that encompasses the inverted repeat. Intrastrand pairing of the entire inverted repeat may then occur to generate the fully extruded cruciform in a single step.

drous radius of the cation present, 26 suggesting that the transition state for the reaction has partial four-way junction character. The larger activation energy for the C-type cruciform extrusion is consistent with a greater degree of helix opening, which would be facilitated by low ionic strength. This large-scale opening is associated with the (A + T)-rich context of the inverted repeat. 2° Indeed, C-type extrusion can be closely approximated in normal DNA sequences if the helix stability is reduced by solvents such as dimethyl formamide27; conversely, if the helical stability of (A + T)-rich DNA is increased with distamycin, C-type cruciforms extrude with kinetic properties that are essentially similar to 24 K. M. Sullivan and D. M. J. Lilley, J. Mol. Biol. 193, 397 (1987). 27 K. M. Sullivan and D. M. J. Lilley, Nucleic Acids Res, 16, 1079 (1988).

164

NONSTANDARD D N A STRUCTURES AND THEIR ANALYSIS

[8]

S-type cruciformsY The (A + T)-rich sequences can be relatively short; a core sequence of 30 bp from ColE1 confers normal C-type extrusion on an adjacent inverted repeat, ~ and we have observed residual effects down to a length of only 12 bp (A. I. H. Murchie and D. M. J. Lilley, unpublished data). The effect can be blocked by a short (G + C)-rich DNA sequence if placed in an intervening position. 2s The efficacy of the (A + T)-rich sequence can be considerably increased by methylation of adenine bases (A. I. H. Murchie and D. M. J. Lilley, unpublished data.) Direct evidence for large-scale opening in the (A + T)-rich DNA has been obtained by two approaches. First, the sequences arc chemically reactive above 25 °29a to probes that react with unpaired bases (see [7] in Volume 212, this series). Second, thermal unwinding of (A + T)-rich sequences has been detected in supercoiled DNA by two-dimensional gel electrophoresis, 3°,3~ and the ColE1 sequences that promote C-type cruciform extrusion exhibit a series of tempcrature-dependcnt transitions (see [5] in Volume 212, this series). In addition, statistical mechanical helixcoil calculations on these sequences indicate a high propensity for cooperative melting, and they demonstrate a correlation between the calculated melting temperature and the experimental temperature of cruciform extrusion. 32 Some sequences undergo cruciform extrusion without a measurable kinetic barrier. Alternating adenine-thymine sequences [(AT)n ] undergo very rapid cruciform formation even at 0 ° ?4 We presume this to reflect the low helix stability of the (AT), sequences themselves, undergoing rapid, cooperative bubble formation and permitting facile extrusion of the cruciform. Structure of the Cruciform A cruciform structure comprises three components: the loops, the stems, and the four-way junction. The stems appear to consist of fairly normal duplex DNA, being cleavable by restriction enzymes, for example. However, one distinction from the rest of the plasmid DNA should be noted: the DNA of the stems is topologically distinct from the remaining DNA in the circular molecule. In other words, the cruciform stems are the only sections of duplex DNA in the molecule that are not subject to superhelical stress. Progress in understanding the structures of the loops 28K. M. Sullivan, A. I. H. Murchie, and D. M. J. Lilley,J. Biol. Chem. 263, 13074(1988). 29j. C. Furlong, K. M. Sullivan, A. I. H. Murchie, G. W. Gough, and D. M. J. Lilley, Biochemistry 28, 2009 (1989). 29aR. Bowater,F. Aboul-ela,and D. M. J. Lilley,Biochemistry 30, 11495(1991). 3oF. S. Lee and W. R. Bauer, Nucleic Acids Res. 13, 1665(1985). 31D. Kowalski, D. A. Natale, and M. J. Eddy,Proc. Natl. Acad. Sci. U.S.A. 85, 9464 (1988). 32F. Schaeffer,E. Yeramian, and D. M. J. Lilley,Biopolymers 28, 1449(1989).

[8]

SUPERCOmED D N A AND CRUCIFOR~t STRUCTURES

165

and the junctions has come from studies of these entities in isolation. It is difficult to study structures that require DNA supercoiling for their continued existence for two reasons. First, a cleavage anywhere within the DNA molecule releases the superhelicity, and the structure is instantly destabilized. Second, supercoiled circles are relatively large molecules for study, making most physical techniques difficult or even inapplicable. There have been a number of studies of hairpin loops in DNA by spectroscopic and thermodynamic methods. Chemical probing of symmetrical loops in cruciforms suggested that the optimal size is around four nucleotides, aa in agreement with an oligonucleotide melting study of the stability of a series of thymine loops of different sizes, a4 However, some sequences are capable of forming a loop of only two unpaired bases, a5 NMR studies of hairpin loop structures of various sequences indicate that there is considerable structure within the loop and that in general the formally unpaired bases of the loop may undergo stacking and hydrogen bonding interactions. 35-as Hairpin structure has been reviewed by van de Ven and Hilbers. a9 The structure of the four-way junction is of considerable interest and importance, as it is formally equivalent to the HoUiday junction, the putative central intermediate of genetic recombination, 4°-~ and there is good evidence for the involvement of a four-way junction in the integrase class of site-specific recombination events. 47-5° DNA junctions are sub33 G. W. Gough, K. M. Sullivan, and D. M. J. Lilley, EMBOJ. 5, 191 (1986). C. A. G. Haasnoot, C. W. Hilbers, G. A. van der Marel, J. H. van Boom, U. C. Singh, N. Pattibiraman, and P. A. Kollman, J. Biomol. Struct. Dyn. 3, 843 (1986). 35 L. P. M. Orbons, A. A. van Beuzekom, and C. Altona, J. Biomol. Struct. Dyn. 4, 965 (1987). 36 L. P. M. Orbons, G. A. van der Marel, J. H. van Boom, and C. Altona, Nucleic Acids Res. 14, 4188 (1986). 37 D. R. Hare and B. R. Reid, Biochemistry 25, 5341 (1986). 3s M. J. J. Blommers, C. A. G. Haasnoot, C. W. Hilbers, J. H. van Boom, and G. A. van der Marel, NATOASISer. Ser. E 133, 78 (1987). 39 F. J. M. van de Ven and C. W. Hilbers Eur. J. Biochem. 178, 1 (1988). 4o R. HoUiday, Genet. Res. 5, 282 (1964). 41 T. R. Broker and I. R. Lehman, J. Mol. Biol. 60, 131 (1971). 42 T. L. Orr-Weaver, J. W. Szostak, and R. J. Rothstein, Proc. Natl. Acad. Sci. U.S.A. 78, 6354 (1981). 43 H. Potter and D. Dressier, Proc. Natl. Acad. Sci. U.S.A. 73, 3000 (1976). H. Potter and D. Dressier, Proc. Natl. Acad. Sci. U.S.A. 75, 3698 (1978). 4s N. Sigal and B. Alberts, J. Mol. Biol. 71, 789 (1972). 46 H. M. Sobell, Proc. Natl. Acad. Sci. U.S.A. 69, 2483 (1972). 47 p. A. Kitts and H. A. Nash, Nature (London) 329, 346 (1987). 4s S. E. Nunes-Dtaby, L. Matsomoto, and A. Landy, Cell (Cambridge, Mass.) 50, 779 (1987). 49 R. Hoess, A. Wierzbicki, and K. Abremski, Proc. Natl. Acad. Sci., U.S.A. 84, 6840 (1987). 50 M. Jayaram, K. L. Crain, R. L. Parsons, and R. M. Harshey, Proc. Natl. Acad. Sci. U.S.A. 85, 7902 (1988).

166

NONSTANDARD D N A STRUCTURES AND THEIR ANALYSIS

[8]

strates for a special class of resolving enzymes, which have been isolated from widespread sources including bacteriophages, 5~,52 yeast, 5a,54 and calf thymus. 55 Stable DNA junctions have been constructed in linear DNA by choosing sequences that are unable to undergo branch migration. 56-5s Gel electrophoretic experiments on a pseudocruciform structure in linear DNA showed that the junction caused a considerable retardation in mobility in polyacrylamide, which was interpreted in terms of a bending or kinking of the DNA at the junction, Ss and this was shown to be dependent on the concentration of cations. 59 As in so many areas of nucleic acid structure, gel electrophoresis has proved to be immensely important in deducing the structure of the four-way DNA junction? s,6°,6~ We used this technique to show that, in the presence of cations, the DNA junction folds into an X-shaped structure, in which the helical arms undergo pairwise stacking to form two quasi-continuous coaxial helices. 6~ Coaxial helical stacking was consistent with the cleavage of junctions by the restriction enzyme MbolI, when binding and cleavage sites were on opposite sides of the point of strand exchange. 62 These stacked arms are rotated (in the manner of opening a pair of scissors) in order to generate the stacked X-structure. Two isomers of the structure are possible depending on which arm is stacked with which, but we have found that for the majority of sequences, one isomer is favored over the other. The structure is consistent with gel electrophoretic experiments, 6! fluorescence resonance energy transfer measurements, 63 enzyme and chemical probing, ~-as and electric birefringence measurements. 6v

51 B. Kemper and M. Garabett, Eur. J. Biochem. 115, 123 (1981). 52 B. de Massey, F. W. Studier, L. Dorgai, F. Appelbanm, and R. A. Weisbcrg, Cold Spring Harbor Symp. Quant. Biol. 49, 715 (1984). 53 L. Symington and R. Kolodner, Proc. Natl. Acad. Sci. U.S.A. 82, 7247 (1985). 54 S. C. West and A. Korner, Proc. Natl. Acad. Sci. U.S.A. 82, 6445 (1985). 55 K. M. Elborough and S. C. West, EMBO J. 9, 2931 (1990). 56 L. R. Bell and B. Byers, Proc. Natl. Acad. Sci. U.S.A. 76, 3445 (1979). 57 N. R. Kallenbach, R.-I. Ma, and N. C. Seeman, Nature (London) 305, 829 (1983). ss G. W. Gough and D. M. J. Lilley, Nature (London) 313, 154 (1985). s9 S. Diekmann and D. Lilley, Nucleic Acids Res. 14, 5765 (1987). 6oj. p. Cooper and P. J. Hagerman, J. Mol. Biol. 198, 711 (1987). el D. R. Duckett, A. I. H. Murehie, S. Diekmann, E. yon Kitzin~, B. Kemper, and D. M. J. LiUey, Cell (Cambridge, Mass.) 55, 79 (1988). 62 A. I. H. Murchie, J. Portugal, and D. M. J. Lilley, EMBO J. 10, 713 (1991). 63 A. I. H. Murchie, R. M. Clegg, E. yon Kitzing, D. R. Duekett, S. Diekmann, and D. M. J. Lilley, Nature (London) 341, 763 (1989). M. E. Churchill, T. D. Tullius, N. R. Kallenbaeh, and N. C. Seeman, Proc. Natl. Acad. Sci. U.S.A. 85, 4653 (1988). 6s M. Lu, Q. Guo, N. C. Seeman, and N. R. Kallenbaeh, J. Biol. Chem. 264, 20851 (1989).

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SUPERCOILED D N A AND CRUCIFORM STRUCTURES

167

On folding of the junction into the stacked X-structure, the reduction of symmetry generates two different kinds of strands in the structure; the continuous strands possess a continuous helical axis, whereas the exchanging strands pass from one stack to the other. Stereochemical considerations suggest that unfavorable steric and electrostatic interactions will be minimized if the X-structure forms a right-handed cross with an antiparallel alignment of continuous strands: the exchanging strands enter and leave the point of strand exchange about the small angle of X. This generates a favorable accommodation of the continuous strands 3' to the point of strand exchange in the major groove of the other helical stack, thereby avoiding steric clash, consistent with the results of DNase I probing studies. 66 The alignment of strands and grooves will be optimal if the small angle of the X-structure is 60 °, and a crystal structure showing similar packing between oligonucleotides has been presented. 6s The stacked Xstructure is illustrated in Fig. 3. Analysis of the structure of a four-way DNA junction using molecular mechanics confirmed that the righthanded, antiparallel stacked X-structure was likely to be the most energetically favorable conformation. 69 The two sides of the X-structure are not equivalent, as the four base pairs at the point of strand exchange are oriented in the same direction. This generates a side that presents the major groove edges and one that presents the minor groove edges of these base pairs, and the two sides have distinct structural properties. It also appears that enzymes are able to distinguish the two sides of the junction; a number of resolving enzymes (e.g., T4 endonuclease VII) selectively cleave on the minor groove side. 7° Detection of Cruciform Structures There are two contrasting ways in which cruciform structures can be detected in a supercoiled circular DNA molecule.

Gel Electrophoresis Formation of a stable cruciform may be detected by the relaxation of a topoisomer, according to Eq. (6), 13'71'72which is revealed as a mobility shift

A. I. H. Murchie, W. A. Carter, J. Portugal, and D. M. J. Lilley, Nucleic Acids Res. 18, 2599 (1990). 67 j. p. Cooper and P. J. Hagerman, Proc. Natl. Acad. Sci. U.S.A. 85, 4653 (1989). 6s y. Timsit, E. Westhof, R. P. P. Fuehs, and D. Moras, Nature (London) 341, 459 (1989). 69 E. von Kitzing, D. M. J. LiUey, and S. Diekmann, Nucleic Acids Res. 18, 2671 (1990). 70 A. Bhattacharyya, A. I. H. Murchie, E. von Kitzing, S. Diekmann, B. Kemper, and D. M. J. Lilley, J. Mol. Biol. 221, 1191 (1991). 71 K. Mizuuchi, M. Mizuuchi, and M. Gellert, J. Mol. Biol. 156, 229 (1982).

168

NONSTANDARD DNA STRUCTURESAND THEIR ANALYSIS

[8]

,

F16. 3. Ribbon representation of the stacked X-structure for the four-way DNA junction.63 The structure is constructed by pairwise stacking of arms to generate quasi-continuous coaxial pairs, with a right-handed rotation of approximately 60 °. The 2-fold symmetric structure contains two kinds of strands; the continuous strands have unbroken axes through the junction, whereas the exchanging strands pass between the two stacked pairs at the point of strand exchange. The two continuous strands have an approximately antiparallel alignment. The arrangement leads to a favorable mutual accommodation of the continuous strands in the major groove of the opposite stacked helical pair, thereby minimizingunfavorable steric and electrostatic interactions. The folded structure of the junction requires cations for stability.

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SUPERCOILED D N A AND CRUCIFORM STRUCTURES

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in an agarose or polyacrylamide gel. This is seen most clearly using a two-dimensional gel,7a where the formation of the cruciform above a threshold level of supercoiling generates a discontinuous "jump." The degree of unwinding on cruciform formation may be measured from the amplitude of the mobility shift, and this coupled with the critical linking difference allows one to calculate the free energy of cruciform formation. The full method is described in [5] in Volume 212, this series. This approach is very powerful and is completely nonperturbing. The drawback of the gel electrophoresis method is that only global changes in the entire circular molecule are observed, and these cannot be interpreted directly in terms of local structure. The gel method shows that there is a structural change somewhere in the molecule, but not where. Frequently the twist change can be correlated with the sequence of the molecule in order to deduce the likely nature of the structural transition undergone.

Probing Enzyme or chemical probes provide a radical alternative to gel electrophoresis methods, and they help to localize the structural change to a particular place in the base sequence. The first demonstration of cruciform formation in natural DNA molecules was based on the cleavage by singlestrand-specific nucleases at the center of inverted repeat sequences when present in supercoiled DNA. 4,5 Probing methods rely on some feature of the structure being recognizably different from the remaining duplex DNA of the molecule, and for a cruciform structure this means either the singlestranded loops or the four-way junction (see above). A summary of some of the probes that have been employed for cruciform structures is given in Table I, and more details may be found in Volume 212 of this series. The potential problem with probes is a kind of uncertainty principle; in order to recognize the modified structure there must be an interaction between probe and DNA, and this might modify the structure under investigation. This is of particular concern for enzyme probes, where the interaction between the substrate and the enzyme binding site might alter the DNA structure. For this reason probing methods should be applied with caution, and the results interpreted with care. However, probes provide the information that gel methods lack, namely, the location of the

72 V. I. Lyamichev, I. G. Panyutin, and M. D. Frank-Kamenetskii, FEBS Lett. 153, 298 (1983). 73 j. C. Wang, L. J. Peck, and K. Becherer, Cold Spring Harbor Syrup. Quant. Biol. 47, 85 (1983).

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NONSTANDARD D N A STRUCTURES AND THEIR ANALYSIS

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TABLE I PROBES USED FOR THE DETECTION AND ANALYSISOF CRUCIFORM STRUCTURESIN

SUPERCOILEDDNA° Enzyme

Target

Special conditions or comments

Refs.

SI nuclease Micrococcal BAL31 P 1 nuclease Mung bean T4 endo VII T7 endo I Restriction Methylases

LOop LOop Loop Loop Loop Junction Junction Loop, junction Loop, junction

LOw pH, Zn2+ required Caz+ required Ca2+ required Range of pH possible Low salt possible Highly specific cleavage Also has single-strand specificity Cleavageinhibited by nonduplex Methylation inhibits subsequent restriction

4,5 b 13 c d e, f 5,52,g 11 25

Reagent

Target

Product

Analysis*

Refs.

Haloacetaldehyde Osmium tetroxide Diethyl pyrocarbonate Glyoxal Bisulfite

Loop A, C Loop T > C Loop A > G Loop G Loop C

S1 or DMS Piperidine, sequence Piperidine, sequence S1 Sequence C to T

i j k l 33

Psoralen

Stem

Etheno adduct cis-Diester Carbethoxylate Etheno adduct Deamination to dU Cross-linking

Cross-linked product

17

° Further details on many of these probes may be found in Volume 212, this series. The references given are the applications of the probes to the study of cruciforms. b D. M. J. Lilley, Cold Spring Harbor Syrup. Quant. Biol. 47, 101 (1983). c D. B. Haniford and D. E. Pulleyblank, Nucleic Acids Res. 13, 4343 (1985). dL. G. Sheflln and D. Kowalski, Nucleic Acids Res. 12, 7087 (1984). • K. Mizuuchi, B. Kemper, J. Hays, and R. A. Weisberg, Cell (Cambridge, Mass.) 29, 357 (1982). .r D. M. J. Lilley and B. Kemper, Cell (Cambridge, Mass.) 36, 413 (1984). s p. Dickie, G. McFadden and A. R. Morgan, J. Biol. Chem. 262, 14826 (1987). hS1 refers to cleavage of the adduct with SI nuclease and DMS (dimcthyl sulfate) to secondary chemical modification. i D. M. J. Lilley, Nucleic Acids Res. 11, 3097 (1983). J D. M. J. Lilley and E. Palecek, EMBO J. 3, 1187 (1984). k j. C. Furlong and D. M. J. Lilley, Nucleic Acids Res. 14, 3995 (1986); P. M. Scholten and A. Nordheim, Nucleic Acids Res. 14, 3981 (1986). i D. M. J. Lilley, in "The Role of Cyclic Nucleic Acid Adducts in Carcinogenesis and Mutagenesis" (B. Singer and H. Bartsch, eds.), p. 83. IARC Publ. 70, Lyon, France, 1986.

[8]

SUPERCOILED DNA At,U>CRUCn:ORMSTRUCTURES

171

structural alteration. For these reasons the best way to proceed is by a combination of methods, using topology along with a number of enzyme and chemical probes. Such comparisons have shown that in the case of cruciform structures the concerns about perturbation by the probes were unfounded, 13 but for other structures this might not be the case. A number of useful enzyme probes are single-strand-specific nucleases, such as S1 or P1 nucleases, that cleave the bases of loop regions more rapidly than duplex DNA. Such cleavage should always be followed by a complete digestion by a restriction enzyme before analysis of the products by gel electrophoresis, so that only cleavage at a specific site is studied. This is necessary because enzymes such as S1 nuclease generate significant background levels of cleavage throughout a supercoiled DNA molecule. It is also important that "single-hit" conditions are employed, since random cleavage anywhere in the molecule releases the supercoiling. Most enzyme probes will function at reduced temperature, and we find that background cleavage elsewhere in the molecule is minimized if the digestion with the nuclease probe is performed at 15 ° or lower. Restriction enzymes H and methylases25 can also be used as probes of cruciform structure, as the loops are refractory to both kinds of enzyme. Enzymes that recognize the fourway junction are the most specific probes of cruciform structure, and their use is the method of choice for many experiments such as measurement of extrusion kinetics. The resolving enzymes, such as T4 endonuclease VII, 5~ exhibit high specificity for cruciform structures, and very low background levels of cleavage are found elsewhere in the molecule, compared to S 1 nuclease, for example. These enzymes were previously available only in very small quantities, restricting their general use, but now the genes for T4 endonuclease VII 74 and T7 endonuclease 175 have been cloned and overexpressed. Most chemical probes react with bases that exhibit single-stranded character in the loops, although some such as osmium tetroxide may react with bases at the junction in the absence of sufficient cations to permit f o l d i n g . 76,77 Many of these probes are discussed in Volume 212 of this series. Once again, some caution is required in the interpretation of results with chemical probes, particularly if only a single probe is employed. The exact nature of the target should be borne in mind, and to describe a chemical probe as single-strand selective may be an oversimplification in some cases. However, if used properly, such probes can be valuable. 74j. Tomaschewski, Ph.D. Thesis, Universittit Bochum, Germany (1988). 75 N. Panayotatos and A. Fontaine, J. Biol. Chem. 262, 11364 (1987). 76j. A. McClellan and D. M. J. Lilley, J. Mol. Biol. 197, 707 (1987). 77D. R. DuckeR, A. I. H. Murchie, and D. M. J. Lilley, E M B O J . 9, 583 (1990).

172

NONSTANDARD D N A STRUCTURES AND THEIR ANALYSIS

[8]

Chemicals such as osmium tetroxide and diethyl pyrocarbonate generate base adducts that are sensitive to alkali and may therefore be analyzed at single-base resolution, generating data of high precision. Others can be converted to the same precision by means of a second chemical modification. 78 Some chemical probes have been used inside cells,79,s° and cruciform formation inside bacteria has recently been demonstrated, using osmium tetroxide and bipyridine to modify the loops of the extruded cruciform.S! Preparation of Supercoiled Plasmid D N A Supercoiled DNA must be prepared and handled with care for two reasons. First, cleavage anywhere within the entire circular molecule completely changes the topology and releases the superhelical constraint. Second, because of their topology, the molecules are more sensitive to damage than normal DNA, and conditions that lower helix stability should be avoided. When preparing DNA for structural studies we do not use preparative methods based on alkali or thermal denaturation, as such vigorous denaturation of the molecules could well lead to the irreversible formation of alternative structures in the DNA. Instead we adopt procedures based on more gentle lysis of cells, followed by isopycnic gradient centrifugation of the supercoiled plasmid DNA in cesium chloride, in the presence of ethidium bromide. This method of separation is preferred for several reasons. First, the conditions are mild. Second, the separation depends on the topology of the DNA, and the method specifically separates constrained from nicked DNA circles. It therefore gives a higher yield of supercoiled DNA than other methods. Third, the DNA is taken from the gradient as positively supercoiled DNA in the presence of a high concentration of an intercalator, and it is free of cruciform and other structures that require negative supercoiling. Subsequent careful removal of the ethidium ions (we use solvent extraction at low temperature) can leave the DNA free of perturbed structures (such as most cruciforms) so that the kinetics of their formation may be studied. The following is the procedure used in our laboratory to prepare supercoiled plasmid DNA for structural studies, including the kinetics of cruci7s y. Kohwi and T. Kohwi-Shigematsu, Proc. Natl. Acad. Sci. U.S.A. 85, 3781 (1988). 79 S. Sasse-Dwight and J. D. Gralla J. Biol. Chem. 264, 8074 (1989). 8o E. Palecek, E. Rasovska, and P. Boublikova, Biochem. Biophys. Res. Commun. 150, 731 (1988). 81 j. A. McClellan, P. Boublikova, E. Palecek, and D. M. J. Lille),, Proc. Natl. Acad. Sci. U.S.A. 87, 8373 (1990).

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SUPERCOILED D N A AND CRUCIFORM STRUCTURES

173

form extrusion. The protocol utilizes pBR322-based plasmids transformed into a c o m m o n laboratory Escherichia coli strain such as HB 101.

Growth and Lysis of Bacterial Cells Exact growth conditions will depend on strain, plasmid, and conditions; the following applies to a pBR322-type plasmid grown in HB101. Prepare 500 ml of M9 medium in a 2-liter culture flask, containing 1.5 g KH2PO4, 3.0 g Na2HPO4, 0.25 g NaC1, and 0.5 g NH4C1, and autoclave. When cool, add the following, using aseptic technique: 2.5 ml of 40% glucose, 25.0 ml of 10% casamino acids, 1.0 ml of 1 mg/ml thiamin, 0.5 ml of 1 M MgSO4, and 2.0 ml of 0.25% thymidine. Any antibiotic required for selection should be added at this stage. Inoculate with 5 ml of a fresh overnight culture of the plasmid-bearing strain, grown in LB medium. Shake the flask with good aeration at 37 ° until the cell density gives an A66o of 0.6 (usually 3 - 4 hr after inoculation). If chloramphenicol amplification is desired, 75 mg chloramphenicol is added at this stage, and shaking continued for a further 16- 18 hr. Longer growth periods can result in lower levels of supercoiling in the extracted plasmid. The cells are collected by centrifugation at 7500 rpm for 10 min at 4 °. The supernatant is decanted into dilute hypochlorite solution. The cellular pellet is resuspended in 7.5 ml of 25% sucrose in 50 m M Tris, pH 8.0, and kept on ice for 10 min. The following additions are made, with constant swirling to ensure good lysis (the vessel is placed on ice for 10 min between each addition). (1) 0.75 ml of 10 mg/ml lysozyme in 25 m M Tris, pH 8.0; (2) 1.5 ml of 250 m M EDTA; (3) 1.5 ml of 5 M NaC1; and (4) 1.5 ml of 10% sodium dodecyl sulfate (SDS). At this point lysis occurs, and the suspension becomes very viscous. The lysate is left for 4 - 2 0 hr on ice.

Isopycnic Gradient Centrifugation of Plasmid DNA The lysate is cleared by centrifugation at 20,000 rpm for 45 rain at 4°; the supernatant is recovered and its volume measured. To this is added 0.97 g of CsC1 and 25/tl of a 10 mggml solution of ethidium bromide, per milliliter of supernatant. The mixture is centrifuged at 10,000 rpm for 10 min, and the clear solution is carefully removed from the dark pellicle and placed in ultracentrifuge tubes. For the fastest density gradient centrifugation we employ a vertical rotor (Beckman VTi 65). The centrifugation is performed at 54,000 rpm at 15 ° for 7 - 16 hr. The (lower) plasmid band is recovered by side puncture (it is usually quite visible in daylight; a UV light source can be employed, but there is a danger of UV-induced damage of supercoiled DNA) and combined in a fresh centrifuge tube, which can be filled if necessary with a solution of 0.97 g/ml CsC1, 250/tg/ml ethidium

174

NONSTANDARD D N A STRUCTURES AND THEIR ANALYSIS

[8]

bromide. This is centrifuged at 54,000 rpm at 15 ° for at least 10 hr. The plasmid DNA is again recovered by side puncture, transferred to a microcentrifuge tube (Eppendorf), and placed on ice.

Preparation of Cruciform-FreeSupercoiledDNA At this stage the DNA is still in the presence of a high concentration of ethidium bromide. As a consequence it is positively supercoiled and free of any structures that are underwound, including cruciforms. To regenerate negatively supercoiled DNA the ethidium bromide must be removed; this must be done using conditions that do not result in the formation of cruciform structures and is therefore done at low temperature throughout, and with avoidance of helix-destabilizing agents. We employ repeated extraction into 1-butanol to remove ethidium bromide, followed by dialysis. An equal volume of l-butanol at 0* is added to the plasmid solution and vortexed. The layers are separated, and the organic (upper) phase removed. A further volume of 1-butanol is added, and the entire procedure repeated 7 times. Care should be taken throughout to maintain a low temperature. It should be noted that the extraction also reduces the volume of the aqueous phase to some extent, and if this becomes excessive precipitation of CsC1 will occur. This can be prevented by restoration of volume. Following extraction of ethidium bromide, the negatively supercoiled DNA is extensively dialyzed against 10 m M Tris, pH 7.5, 0. l m M EDTA at 4°, in order to remove CsC1. On removal from the dialysis sac the DNA is not manipulated further, and it can be used directly for kinetic and other measurements. The yield of DNA depends on the plasmids and strains used, but typical yields range from 200/zg to 2 mg DNA from 1 liter of culture. The concentration after the second gradient is normally in the range 200-1000 #g/ml. The DNA should be over 95% supercoiled at the end of the preparation. We store the supercoiled DNA in aliquots at -- 7 0 ° and gently thaw the frozen DNA at 4 ° just before use.

Preparation of Single Topoisomeric Species For some studies it is useful to work with preparations of a circular plasmid that contain one single topoisomeric species or a narrow Gaussian distribution of topoisomers. For an example, see Lilley and Hallam? 3 Preparation of the latter is described in [5] in Volume 212, this series. The high resolving power of agarose gels is used for the separation of topoisomers, and the individual species are excised and electroeluted.

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SUPERCOILED D N A AND CRUCIFORM STRUCTURES

175

A topoisomer distribution centered on the linking difference of interest must first be prepared by relaxation of supercoiled plasmid with topoisomerase I in the presence of an appropriate concentration of ethidium bromide (see [5] in Volume 212, this series). Ten to sixty micrograms of DNA is relaxed and loaded on to a 1 cm thick agarose gel in 90 m M Tris-borate, pH 8.3, 1 m M EDTA (TBE buffer). A single lane of 2 cm width can take 12/lg of DNA without overloading. The 25 cm long gel is electrophoresed until the supercoiled DNA has traveled about 80% of the length of the gel (the speed of migration will depend on the size of plasmid, and the time of eleetrophoresis will have to be determined in a trial experiment). The bands are visualized by staining in 1 pg/ml ethidium bromide and illumination on a transilluminator at 300 nm. We find that a glass plate inserted between the transilluminator and the gel helps to avoid DNA breakage by the UV fight, thereby improving the integrity of the topoisomers. The required topoisomers are excised with a scalpel and electroeluted. This DNA is sufficiently pure for most purposes but can be further purified if required, using isopycnic centrifugation. For this we employ a small-angle rotor in a Beckman TL100 centrifuge, taking a tube of 1 ml volume. We find that quantities of DNA as low as 1/lg can be successfully purified using this method. R a t e M e a s u r e m e n t s of Cruciform Extrusion In general, cruciform extrusion is highly temperature dependent because of large enthalpies of activation. Hence conditions can be chosen under which the process is relatively slow, that is, on a time scale of minutes to hours. Moreover, the temperature dependence can be exploited as a means of starting and ending the extrusion process at will; thus, despite the use of rather unsophisticated techniques, it is possible to study extrusion during time intervals down to about 30 see. Two kinds of kinetic experiments may be performed: (1) To examine the time course of cruciform extrusion under a fixed set of conditions, a sample of the plasmid is incubated at a given temperature and buffer conditions, and aliquots are removed at various times for subsequent analysis of cruciform extrusion. (2) For analysis of cruciform extrusion as a function of conditions, a set of samples can be incubated for a fixed period, while varying a particular parameter such as sodium ion concentration. At the end of the period all samples are analyzed for cruciform extrusion. The outline of the procedure for kinetic measurements can be summarized as follows:

Preparation of Cruciform-Free DNA. DNA preparation is performed as above. At the beginning of the kinetic procedure the DNA is diluted into the required buffer at 4 °. No extrusion occurs under these conditions.

176

NONSTANDARD D N A STRUCTURES AND THEIR ANALYSIS

[8]

Extrusion Interval. The supercoiled DNA sample at a concentration of about 30/~g/ml is then placed in a water bath at the required temperature for a certain time, in the buffer conditions (composition, pH, ionic strength) appropriate to the experiment. Aliquots may be removed at various times from a sample incubated at the fixed temperature. Because of the high temperature dependence of these reactions, accurate and precise temperature control is essential. We routinely use water baths that can be set with a precision of 0.01 °. Termination of Extrusion. The sample or aliquot is rapidly transferred to ice to stop any cruciform extrusion. After 5 rain, concentrated reaction buffer is added, and the sample is left for a further period. Assay of Cruciform Formation. The assay of cruciform formation is carried out using a nuclease that selectively cleaves the cruciform structure. In the majority of published studies S1 nuclease has been used to cleave the cruciform loops, but the advent of cloned resolving enzymes makes these the reagents of choice owing to their much higher specificity for the cruciform. Both types of enzymes can be used quite successfully at 15 °, under which conditions no cruciform extrusion occurs during the incubation (at least, for the great majority of sequences). At the end of the incubation, the DNA is ethanol precipitated, redissolved, and cleaved to completion with a restriction enzyme. The DNA is then analyzed by gel electrophoresis. It is best to choose a restriction enzyme that cleaves once only in the plasmid, at a site relatively close to the inverted repeat studied. Typical results of a time course of cruciform extrusion are given in Fig. 4. The upper band (band F) is the full-length linear DNA, which corresponds to restriction cleavage of DNA that had not been cleaved at a cruciform structure. The second band (band C) migrating ahead of the full-length species is that arising from an initial cleavage by the nuclease (S1 nuclease in this example) at the cruciform, followed by restriction cleavage; this band is diagnostic for the presence of the cruciform, and its intensity is related to the amount of cruciform extrusion that occurred during the incubation step. This may be quantified by densitometry. If T4 endonuclease VII is used to assay for cruciform extrusion, the relative intensity of band C can be taken as a good estimate of the amount of cruciform extrusion in absolute terms. However this is not the case for S 1 and other less specific nucleases. With these nucleases there is too much nonspecific cleavage elsewhere in the molecule. Under these conditions, the intensity of band C is only proportional to the extent of extrusion, and one obtains only a relative measure of the degree of cruciform extrusion. However, this is adequate for the measurement of rate constants.

[8]

SUPERCOILED DNA AND CRUCIFORMSTRUCTURES

0

1

2

3

6

10

15

25

177

min

~"

F

~"

C

FIG. 4. Example of a time course for the extrusion of a cruciform in a supercoiled plasmid. The plasmid studied is pIRbke8, in which adenine bases at the center of the inverted repeat have been Nt-methylated using EcoRI methylase and S-adenosylmethionine. 25 Cruciform extrusion proceeds by the S-type mechanism. Cruciform-free supercoiled DNA was incubated at 37* in 10 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 50 mM NaC1. Aliquots were removed at the times indicated and stored at 0". They were then digested with 2 units of S1 nuclease in 50 mM sodium acetate, pH 4.6, 50 mM NaCI, 1 mM ZnCI2 at 15" for 30 rain, before complete cleavage by HindlII. The DNA was electrophoresed in 1% agarose, stained with 1 #g/ml ethidium bromide, and photographed under UV illumination. Two bands can be distinguished; band F is full-length plasmid, and band C is the shortened species arising from S1 nuclease cleavage at the loop of the extruded cruciform. The relative extent of extrusion may be estimated by deusitometry as the ratio of inteusities C/(F + C).

Cruciform extrusion is a unimolecular process represented by DukDx

where D u is unextruded plasmid DNA and Dx is cruciform-containing DNA. This is a first-order process that obeys the equation d [ D J = - k I l : Y d dt

(7)

where D~ is the concentration of unextruded DNA at time t. A time course of the progress of cruciform extrusion is presented in Fig. 5 as a semilogarithmic plot, from the slope of which is obtained the rate constant (k) for the process. The half-time for the reaction may be calculated as

tl/2 = 0.69/k

(8)

178

NONSTANDARD DNA STRUCTURESAND THEIR ANALYSIS

[8]

A 1,2 '

1+In (unextruded/

0.8

total DNA) 0.4

0.0

o

;

i

;

4

Time (rain)

B 1.00

1 +In (unextruded/

0.90

total DNA)

~16plRbke 8

0.80

0.70

i

;

;

a

Time (hours) FIG. 5. Rates of cruciform extrusion for inverted repeats of related sequence, as shown by progress plots of ln(unextruded fraction) against time for four closely related plasmids. The inverted repeat sequences are all based on that of pIRbke8, with either one or two mutations in the repeat unit23;these plasmids extrude by the S-type mechanism, requiring the presence of salt to allow extrusion to proceed. Unextruded plasmid DNA was incubated at 37 ° in 10 mM Tris, pH 7.5, 50 mM NaCI, and aliquots were removed at different times and placed on ice. These were later digested with either (A) SI nuclease or (B) T7 endonuclease I, followed by complete cleavage with HindIII. Note the very different time scales for extrusion for the different inverted repeat sequences. Under these experimental conditions the following reaction half-times were measured: pIRbke8, 70 rain; pIRbke8A, 6 rain; pIRbkeST, 17 rain; plRbke 15C16C, 1649 min. Overall it was observed that the rate of extrusion within this series of sequences varied by a factor of almost 2000.

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SUPERCOILED D N A AND CRUCIFORM STRUCTURES

179

A -6 -7" -6.

C "9"

-10"

-11 3.322

" 3.333

" 3.344

" 3.355

IO00/T

B -6' -7' Jg e-6.

-9"

-10

3.,8

3.;,2

3.;e

3.;o

10001T

FIG. 6. Examples of Arrhenius plots of the temperature dependence of cruciform extrusion for typical C-type and S-type sequences. (A) Temperature dependence of the extn~on of a C-type plasmid pIRxke/col. Rate constants (k) were measured in l0 m M Tris-HC1, pH 7.5, 0.1 m M EDTA as a function of the absolute temperature (T). From these data an activation energy of 215 kcal tool-~ was calculated. (B) Temperature dependence of extrusion of an S-type plasmid pIRxke/vec, in l0 m M Tris-HC1, pH 7.5, 50 m M NaCI. An activation energy of 52 kcal tool-I was calculated. The inverted repeat sequences of pIRxke/col and pIRxke/ vcc are identical; the plasmids differ in the sequences that flank the inverted repeats. In the case of pIRxke/col the contextual sequences are (A + T)-rich sequences derived from the plasmid ColEl, whereas for pIRxkc/vec the xke inverted repeat is embedded in pBR322 sequences of normal base composition. 2°

180

NONSTANDARD D N A STRUCTURES AND THEIR ANALYSIS

[9]

The temperature dependence of cruciform extrusion is well described by the Arrhenius equation, k = A e-r~Rr

(9)

where E, is the Arrhenius activation energy, R the gas constant, T the absolute temperature, and A the temperature-independent, preexponential factor. Good straight lines have been obtained when rate constants measured at different temperatures are plotted against reciprocal temperature, as shown in Fig. 6. The slope of the line is -E/R, from which the Arrhenius activation energy is calculated.

[9] P r o t o n a t e d

DNA Structures

By MAXIM D. FRANK-KAMENETSKII

DNA Protonation Canonical B-DNA has no strong protonation sites, and because of this it does not pick up protons with the exception of totally nonphysiological conditions of extremely low pH. Single-stranded DNA has stronger protonation sites, but it is protonated only below pH 4.3, the pK value for the N-3 position of free cytosines. However, this does not mean that protonation is an insignificant phenomenon for DNA. Some nonorthodox DNA structures, first of all those which include DNA triplexes, provide very strong protonation sites with pK values above 7, which means that these structures are protonated when formed under normal pH conditions. The best studied examples of protonated DNA structures are H-DNA and intermolecular triplexes. Both types of unusual structures have a common element, the triplex, which carries the protonation sites. In DNA triplexes the protonation site is created because of formation of C. G*C + base triads. In this triad, the usual Watson-Crick C . G pair is supplemented by protonated cytosine, which is attached to guanine via the Hoogsteen mode of binding. Not all triplexes are necessarily protonated, and protonated triplexes do not exhaust possible protonated structures. For example, the protonated C.C + noncanonical base pair is another plausible candidate to be an element of an unusual DNA structure. Under neutral and slightly acid conditions (above pH 5), the triplex remains the major element of the protonation structures detected to date. METHODS IN ENZYMOLOGY, VOL. 211

Englishtranslationoat~isht © 1992by A~lemic Press,Inc. All rightsof reproductionin any formreserved.