TRANSITION FROM RIGHT-HANDED TO LEFT-HANDED DNA

GASPAR BANFALVI

Institute of Biochemistry Depar tment I Semmelweis University Medical School 1444 Budapest , Hungary

Introduction

Solomon's knot DNA

Local torsional strain of DNA

Formation of left-handed-loops

Model building of DNA proved to be an important step in the elucidation of the three dimensional structure of DNA.~ However, ball-and-stick models and space-filling models fail to visualize tertiary changes in DNA structure as the introduction of a swivel is likely to destroy the model itself. Theoretically it would be possible to predict topological changes of DNA structures with the aid of computers. Calculations must take into account not only nucleotide sequence and minimum-free energy conformation of DNA based on covalent, electrostatic, van der Waals and steric interactions but also the free energy represented by the torsional strain of superhelicity. Because of the complexity of short- range and long-range factors, some of which may even be unknown, attempts toward this end remained unsuccessful.

Solomon's knot DNA is a simple version of the Watson-Crick model 2 which has the advantage from the educational point of view of being extremely inexpensive to construct and of being very robust in the hands of students. Nevertheless it may be used to illustrate some very important principles. The circularization of such a linear macram6 DNA allows a clear demonstration of superhelicai structures. 3 Topoisomers which are generated by the catalytic activities of topoisomerases 4"5 can be distinguished by their linking numbers that specify the number of times two DNA strands are intertwined. 6"7 The free energy of superhelix formation generated by the torsional strain of DNA can be calculated by a relatively simple equation. 8

By turning the circularized macram6 DNA molecule (Fig la) to the left, the revolution generates positive and negative supercoils without appreciable distortion of the helical structure, since the structural constraint is distributed evenly along the molecule (Fig lb). There is no satisfactory explanation as to why supercoils are formed generally in this direction and not to the right in nature. When we twist the macram6 DNA to the right the torsional constraint is not distributed evenly in the molecule. The local stress generated by right-handed twists decreases the number of helical turns in front of the twist and increases the helicity behind the twisting point (Fig lc). Thus it appears as if the torsional rigidity of the DNA backbone were different against right- and left-handed turns. This could explain why supercoils are formed preferentially to the left-hand direction.

In strand separation processes such as DNA replication, transcription and recombi- nation, the left-handed torsional strain generated by helicases is located to only a relatively small portion of the genome. Helicases move along double-stranded DNA by a wedge-like action to unwind DNA. 9 Forked DNA structures are formed by the concerted action of a set of enzymes. The local stress of melting could be relieved if the whole DNA molecule were allowed to rotate. This is t~nlikely to happen because of the remarkable length of DNA in cells. Moreover, in bacterial DNA is usually attached to an infolding of cell membrane, the mesosome. The unwinding in front of the fork leads to the formation of a positive supertwist (Fig 2).

Local torsional strain may not only melt the duplex DNA but also initiate the formation of left-handed loops without strand separation or strand breaks (Fig 3). Right-handed (B) and left-handed DNAs have quite different conformations. In right-handed DNA the sugar-phosphate backbone is much more flexible and can exist in a very large number of conformations because each single bond in the backbone has a certain freedom of wobble. Although the most stable conformation of macram6 DNA is the B-form on account of the topology of the Solomon's knot, the left-handed-conformation can be "induced" by twisting a segment of the DNA helix into a left-handed configuration. Left-handed DNAs, on the other hand, show one or more rigid kinks contributed by negative loops (Fig 3. a, b, c). This cis configuration of negative loops produces bends in the DNA backbone, whereas right-handed DNA corresponds to a trans configuration.

BIOCHEMICAL EDUCATION 14(1) 1986

Figure 1

b

d~

Demonstration of local distortion of duplex DNA. (a) Linear double-stranded DNA was 'synthesized' using 16 m of 3 mm nylon laundry rope ~ and was circularized with plastic couplers affixed to the ends of the linear molecule. ~ (b) Circular macramd DNA was twisted in the opposite direction of the helical turns ie to the left. One negative (left-handed) and one positive (right-handed) supercoils were formed. (c) Circular macram~ DNA was twisted in the direction of helical turns ie to the right. Increased helicity (<), reduced helicity (>) of duplex DNA.

Figure 2 Positive supercoiling generated by strand separation. Circular DNA was denatured at the region indicated by the arrow.

Figure 3 Demonstration of left-handed loops. Circular DNA was denatured at a small region and turned into negative loop(s). Introduction of (a) one, (b) two and (c) three negative loops.

The formation of left-handed D N A needs strong base-pairing primarily at GC-rich sequences where C residues are methylated. Methylated bases arise by postreplicative

• . C G . . modification at . . . . GC sequences of genomic DNA. In higher plants and animals the

majori ty of . . C G . . dinucleotides are modified by methylation. The methyl group is attached to the 5-carbon a tom of cytosine in poly(dC-dG) which stabilizes the Z - D N A

B I O C H E M I C A L E D U C A T I O N 1 4 ( 1 ) 1 9 8 6

Figure 4

Formation of cruciform and hairpin structures

conformation over B-DNA.I° Methyl groups do not affect base-pairing and the hydrogen bonds of 5-methylcytosine with guanine are equivalent to those formed by cytosine. Methylation of guanine at C-6 has been shown to cause mispairing with uracil ~t and is therefore a rare event in cells.

Model experiments with macram6 DNA indicate that at least 20 base pairs are involved in the formation of a left-handed loop including two tetranucleotide junctions between right-handed and left-handed helical segments and 12 base pairs forming the negative loop (Fig 4). The left-handed loop is slightly funnel-shaped, not quite symmetrical with 12 nucleotides at the inside circle and 13 nucleotides at the outer contour. Negative loops of less than 12 base pairs are probably not formed and would snap back to their right-handed counterparts. The angle of the helical axis of the left-handed DNA is twisted by at least 25 degrees from the helical axis of the right-handed DNA. The two axes are roughly 25/~ apart assuming a transition of 20 nucleotides as shown in the diagram (Fig 4).

Diagrammatic structure of left-handed DNA. Ribbons symbolise sugar-phosphate back- bones held together by base pairs. The horizontal line indicates the fibre axis of right-handed DNA. The axis of left-handed DNA is marked by the slanting line.

Alternating dC-dG sequences are self complementary, ie they have inverted repeat symmetry and are able to form hairpin loops or cruciforms. If a longer region of DNA containing inverted repeats turns into left-handed structure, two or more negative loops are formed. In the process with macram6 DNA, we found that the introduction of negative loops, cruciform or hairpin structures is paralleled by the reduction of negative superhelical turns and by the increase of positive supercoils (Fig 5). This model experiment suggests that the tcansition may serve the purpose of conversion of negative supercoiled structure to positive supercoils without excessive strand separation or strand breaks.

Figure 5 Nondisruptive transition from negative to positive supercoiled structure involving cruciform and hairpin formation. Negative supercoiled DNA (a) was first turned to linear circular DNA by the introduction of a cruciform structure (b) then to positive supercoiled DNA introducing a hairpin structure (c).

In conclusion model experiments with macram6 DNA indicate that:

(1) Right-handed (B-form) and left-handed DNAs can exist in close proxinaity in addition to the already known coexistence of other structural forms such as A, B and B'. (2) The transition is restricted to a small portion of DNA consisting of at least 20 nucleotides to provide a relatively stable left-handed conformation that involves 12

BIOCHEMICAL EDUCATION 14(1) 1986

10

Acknowledgement

References

nucleotide base pairs forming the negative loop and two junction regions (4 nucleotides each) flanked by right-handed DNA. The size and number of loops may vary. The axis of the left-handed loop deviates about 25 degrees and is roughly 25/~ apart from the helical axis of the right-handed DNA. (3) Energetically and sterically a non-disruptive transition induced by local torsional strain seems to be feasible. Transition reduces the number of negative supercoils and increases those of the positive ones. The transition seems to preserve the covalently closed structure of D N A , may serve the maintenance of free energy of supertwisting, the regulation of superhelicity, the activation and inactivation of genes.

The transition from right-handed to left-handed D N A shown by these model experiments is consistent with the experimental data of others. The transition for ( d C - d G ) , inserts was found in supercoiled plasmids, where n = 16, 13 and 5 also was evaluated. 12 Physical, topological and enzymatical evidence suggests that the junction region is small, partially single-stranded and located within non-alternating d C - d G sequences. 13-16 The B - Z transition is sensitive to the salt concentration and C-5 methylation with d C - d G blocks, such that decreasing salt concentrations paralelled by the increase of methylation allow the transition at lower negative superhelical densities.13

The conformational change is likely to have a major impact on the overall topology of the genome. Although the physiological consequences of this transition are not well understood, it is reasonable to regard the transition as a topological sign that is involved in the genetic regulatory mechanism.

I thank Professor F Antoni and H Paulus for the critical reading of the manuscript.

t Watson, J D and Crick, F H C (1953) Nature, 171,737-738 2Fieldhouse, J (1981) Biochemical Education 9, 88 3Banfalvi, G (1984) Biochemical Education 12, 155-156 4Cozzarelli, N R (1980) Science 207,935-960 5Gellert, M, Mizuuchi, K, O'Dea, M H and Nash, H A (1976) Proc Nail Acad Sci USA 73, 3872-3876 6Crick, F H C (1976) Proc Natl Acad Sci USA 73, 2639-2643 7Wang, J C (1983) in DNA Makes RNA Makes Protein edited by Hunt, T, Prentis S and Tooze J, Elsevier Biomedical Press, Amsterdam

8Horowitz, D S and Wang, J C (1984) J Mol Biol 173, 75-91 9Abdel-Monem, M, Lauppe, H F, Kartenbeck. J, Dtirwald, H and Hoffm'ann-Berling, H (1977) EurJ Biochem 110, 667-685

t°Behe, M and Felsenfeld, G (1981) Proc NatIAcad Sci USA 78, 1619-1623 IIGerchman, L L and Lundlum, D B (1973) Biochim Biophys Acta 308, 310-316 t2Singleton, C K, Klysik, J and Wells, R D (1983) Proc Natl Acad Sci USA 80, 2447-2451 13Singleton, C K, Klysik, J, Stirdivant, S M and Wells, R D (1982) Nature 299, 312-316 t4peck, L J, Nordheim, A, Rich, A and Wang, J C (1982) Proc Natl Acad Sci USA 79, 4560-4564 15Klysik, J, Stirdivant, S M, Larson, J E, Hart, P A and Wells, R D (1981) Nature 290, 672-677 ~6Wartell, R M, Klysik, J, Hillen, W and Wells, R D (1982) Proc Natl Acad Sci USA 79, 2549-2553

Journals Available

An American profesor is willing to give away the following journals: Journal o f Bacteriology from 1967 to 1985 bound through 1975, Journal o f Biological Chemistry from 1964 to 1985, bound through 1975, Biochemistry from 1962 to 1985, bound through 1975, Proceedings o f the National Academy o f Sciences from 1962 to 1985, bound through 1972, Federation Proceedings o f the American Society o f Biological Chemistry from 1965 to 1985, bound through 1975. Any library, depart- ment of biochemistry, or individual interested and prepared to pay the costs of crating and shipping one or all of these journals, should write to Professor F Vella, Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada S7N 0W0.

Molecular Biology of the Cell

Garland Publishing is soliciting original problems to be used in a companion Prob lems text that will be published along with the second edition of Molecular Biology o f the Cell by Alberts, et al.

If you are interested in submitting problems, please send your name, position, and mailing ~address to: Dr John H Wilson, Depar tment of Biochemistry, Bayior College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA who will send detailed instructions.

BIOCHEMICAL EDUCATION 14(1) 1986