Organization of DNA in chromatin ( chromatin structure / intercalation / DNA - kinking )

Conformational changes in DNA that accompany drug intercalation have led us to ask if DNA first bends or "kinks" to accept an intercalative drug or dye. Kinking is made possible by altering the normal C2' endo deoxyribose sugar ring puckering in B DNA to a mixed sugarpuckering pattern of the type C3' endo (3'-5') C2' endo and partially unstacking base-pairs. A kinking scheme such as this would require minimal stereochemical rearrangement and would also involve small energies. This has prompted us to ask more generally if a conformational change such as this could be used by proteins in their interactions with nucleic acids. In this parr we describe an interesting superhelical DNA structure formed by kinking DNA every 10 base-pairs. The structure may be used in the organization of DNA in chromatin. The organization of DNA in chromatin is a subject that has attracted growing interest in recent years (for a review, see Elgin and Weintraub, ref. 1). It is generally agreed that chromatin consists of a linear arrangement of bead-like structures (called v bodies) that contain DNA and histones (2). The exact diameter of the bead is uncertain, but is probably in the order of 100 A. Each bead is thought to contain two sets of four different histones [i.e., 2(H-2a, H-2b, H-3, H-4)] complexed with about 170 base-pairs of DNA and an additional histone (i.e., H-1) complexed with about 40 base-pairs (3, 4). This DNA is folded to about one-seventh its length, a value deduced from electron microscopy measurements of minichromosomes of simian virus 40 (SV40) and adenovirus 2 (5, 6). The exact manner in which DNA is folded within the i body is not known. Noll has shown that DNase I digestion of chromatin liberates DNA fragments 10, 20, 30, 40, ... up to 200 bases long (7). This suggests that the DNA lies on the outer surface of the v body and that some structural feature of DNA related to its helical periodicity is recognized and cleaved by the enzyme. Crick and Klug (8) have advanced a specific hypothesis to explain the arrangement of DNA in chromatin. They postulate that DNA is wound around the outer surface of the histone core not by continuously deforming DNA, but by kinking DNA every 20 base-pairs. In their scheme, kinking is accomplished by unstacking base-pairs and altering the sugar-phosphate backbone from its normal gauche-gauche conformation to a gauche-trans conformation. This allows helical sections above and below the kink to come apart and form an angle of 980 between their helical axes. In their model, kinking imparts a small negative twist to DNA, reducing the twist angle from 360 to about 15-200 at the kink. This gives rise to a left-handed (kinked) toroidal helix when DNA is complexed with histone, a structure that subsequently could be detected as a right-handed interwound superhelix in histone-free circular DNA molecules (9). The Crick-Klug stereochemical kinking scheme predicts eight kinks per 170 base-pairs and this gives rise to a left-handed kinked toroidal helix with a diameter of about 90 A that contains Abbreviation: SV40, simian virus 40. somewhat more than two turns per v body. Although their scheme is satisfactory from the stereochemical point of view [even though their sugar-phosphate backbone conformation is not one of the preferred conformations currently listed (10, 11)], we would like to suggest an alternative stereochemical scheme for kinking DNA that results in much the same consequences as regards histone-DNA interaction. The scheme uses our current ideas about drug intercalation into DNA. Stereochemistry of drug intercalation We have recently determined the three-dimensional structures of two ethidium: dinucleoside monophosphate crystalline complexes [ethidium: 5-iodouridylyl(3'-5')adenosine (12-14) and ethidium: 5-iodocytidylyl(3'-5')guanosine (15, 16)] and one 9-aminoacridine: dinucleoside monophosphate crystalline complex [9-aminoacridine: 5-iodocytidylyl(3'-5')guanosine (17, *)] by x-ray crystallography. All three structures demonstrate drug intercalation into miniature Watson-Crick double helices. Features common to these structures are a gauche-gauche sugar-phosphate backbone conformation with altered glycosidic torsional angles (these will be described in detail below) and the following pattern of ribose sugar ring puckering at the intercalation site: C3' endo (3'-5') C2' endo. These conformational changes permit base-pairs to separate 6.8 A and give rise to the observed twist angle between base-pairs above and below the intercalative drug or dye (estimated in these studies to be-between 8 and 10°) as well as to a common relative base-pair orientation as defined by the positions of the glycosidic bonds. We have used this stereochemical information to understand the general nature of intercalative drug binding to DNA. This is shown in Fig. lB and D. To construct the ethidium-DNA binding model, we have added B DNA to both sides of the intercalated dinucleoside monophosphate. This is done easily and without steric difficulty. An important realization that immediately emerges is the concept that drug intercalation requires a helical screw axis dislocation in DNA; our model therefore differs in a fundamental way from other models of intercalation recently proposed (18, 19). We estimate that helical axes for B DNA above and below ethidium intercalation are displaced by about +1.0 A. Base-pairs in the immediate region of intercalation are twisted by 100 (this value has been estimated by projecting the interglycosidic carbon vectors on a common plane and then measuring the angle between them). This gives rise to an angular unwinding of -26° at the immediate site of drug intercalation. We have also observed that intercalated base-pairs are tilted relative to one another by about 8° in both ethidium crystal structures. This results in a small residual "kink" of 60 at the intercalation site, and has been included in our ethidium-DNA binding model (Fig. ID). *T. D. Sakore, S. C. Jain, C. C. Tsai, and H. M. Sobell, manuscript in