Facilitating structural transitions in DNA.

A t one time, the structure of DNA was blissfully simple. It was elegant, regular, and universal—quite unlike the structures of proteins, which were complicated, highly varied, and full of peculiarities consistent with their multifarious functions. DNA was a plectonemic double helix of 10 nucleotide pairs per turn (a nice round number) containing planar base pairs stacked neatly perpendicular to the helix axis and, of course, capable of accommodating any conceivable sequence of nucleotides within its regular structure so that it could encapsulate any kind of biologically meaningful sequence information within its consummate regularity (1). This simple and unifying concept was so compelling, and the imaginative representations printed in textbooks or devised for the media were so visually attractive, that the ‘‘ideal’’ B-form helix dominated the thinking of molecular biologists in the early years and has probably done more than any other single icon to attract brilliant students to venture into biology. But it could not last. Even at the outset it was known that fibers of DNA would give the relatively fuzzy B-form x-ray diffraction pattern at 92% relative humidity, supposed to be the biologically relevant condition, but would switch reversibly to a more crystalline state (the A-form) at 75% relative humidity, characterized by sharper reflections and altered helical parameters like 11 tilted base pairs per turn (2, 3). It was also known that binding of aminoacridines such as prof lavine to DNA would modify the B-form pattern and destroy its helical regularity, although not the perpendicular stacking of the base pairs, and thus the intercalation hypothesis was born (4). About this time, as a Ph.D. student in Cambridge working on ethidium bromide among other things, it seemed to me that ethidium ought to be a better, or at least cleaner, intercalator than proflavine. I enlisted the help of Watson Fuller, then working in Maurice Wilkins’ lab at King’s College London, to try x-ray diffraction on ethidium–DNA complexes (5). We thought that ethidium ought to impede the B7 A transition (which it does, actually), but our first experiments were a spectacular failure. Fibers precipitated from an ethidium–DNA complex underwent the B3A transition on lowering the relative humidity from 92 to 75% as if nothing had happened, and vice versa, but the control fibers—DNA alone that had never seen the drug—did not. Watson reported this disappointing result, with a characteristically jokey final comment, in a letter dated 5 March 1964: . . . . ‘‘As you see the [ethidium] complex behaved as normal Na DNA and the diffraction patterns are indistinguishable from those of normal Na DNA. . . [several notional explanations]. . . One thing—the DNA control did not undergo the A3 B transition (I tried a few times). This does happen from time to time and is probably not important. However, it may be that the ethidium makes the A to B transition easier in some peculiar way: How about a Paper entitled Ethidium Bromide—a molecular grease?’’ I mention this tale because it could well be the first time, albeit erroneously, that anyone advanced the idea that drugs might be able to induce or facilitate long-range structural transitions in DNA. Another current of thinking that can be traced back to early days is the notion that the biological function of DNA, and in particular differential gene expression, might actually require the molecule to adopt different helical states. How else could regulators quickly locate and identify such elements as startystop signals in the huge length of DNA present in a cell nucleus? Searching for sequence-dependent structural variations led to the discovery of an impressive variety of odd things that DNA can do under defined conditions, often related to bending, although for some time at least the base pairing and right-handed helical screw sense remained inviolate. Not for long. One of the biggest upsets to orthodoxy was the discovery of Z-DNA, the lefthanded double-helical conformation with a dinucleotide repeat reported in 1979–1980 (6, 7). And the last bastion crumbled just a few years later when it was found that Watson–Crick base pairs could be induced to flip into the long-outlawed Hoogsteen pairing configuration (8) by binding of bisintercalating quinoxaline antibiotics (9, 10). Add to that the recent excitement generated by G-quadruplex structures adopted by guanine-rich sequences present in the telomeres of mammalian chromosomes (11), and you could be forgiven for thinking that nothing is sacred nowadays. However, it would be foolish to dismiss even outrageous noncanonical structures as mere aberrations or artifacts; Nature has a remarkable way of using rare phenomena to perform biological roles. In many of these discoveries, the binding of drugs played a significant part, as I have indicated. Interestingly, the x-ray crystallographic evidence for a left-handed helical form of poly (dGzdC) at high salt concentration was foreshadowed by the remarkable finding of Pohl, Jovin, and colleagues (12) that ethidium serves as a powerful cooperative allosteric effector modulating the structure of this artificial DNA by virtue of its huge preference for binding to the righthanded double-helical conformation preferred at low salt concentration as opposed to the ‘‘L-form’’ preferred in the presence of high salt. A ‘‘molecular grease’’ indeed? No, because the drug drives the left-handed Z-form back to B-form (or, more accurately, an intercalated right-handed form) because its binding to the polynucleotide duplex perturbs the equilibrium between the two forms at high salt in favor of the latter. A ‘‘grease’’ would act like an enzyme and merely hasten the attainment of thermodynamic equilibrium. Against this background, the report of Qu, Chaires, and colleagues (13) in this issue of PNAS is of classic significance. Starting from the observation that the widely used antitumor antibiotic (1)-daunorubicin (daunomycin) appears well tailored to bind intercalatively to right-handed B-form DNA, the authors decided to synthesize its optical antipode (2)-daunorubicin in the expectation that it might bind selectively to left-handed double-helical DNA. The synthesis of the enantiomer, code-named WP900, was no easy undertaking; it required some 37 steps. Qu et al. first used an ingenious method to determine whether natural daunorubicin and WP900 might bind to double helices of opposite handedness by mixing solutions of the ligands in equal proportions and then seeing which isomer would predominate after dialysis against a sample of poly(dGzdC) in either the right-handed (low salt) or left-handed