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We describe and characterize a complex reaction that catalyzes DNA supercoiling and chromatin assembly in vitro. A Xenopus oocyte extract supplemented with ATP and Mg++ converts DNA circles into minichromosomes that display a native, 200 bp periodicity. When supercoiled DNA is added to this extract it undergoes a time-dependent series of topological changes, which precisely mimic those found when the DNA is microinjected into oocytes. As judged by the conformation of the subsequently deproteinized DNA, the supercoiled DNA is first relaxed, in a reaction that takes 4 min, and then it is resupercoiled in a slower process that takes 4 hr. The relaxation is partially inhibited by EDTA, to an extent that suggests that that it is catalyzed by a type I DNA topoisomerase. The resupercoiling , on the other hand, requires ATP and Mg++, is completely inhibited by EDTA, and is inhibited by novobiocin in a manner that suggests it is catalyzed by a type II DNA topoisomerase. These findings, and the ones reported in the preceding paper ( Ryoji and Worcel , 1984), lead us to propose that chromatin assembly is an active, ATP-driven process.

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Recent observations suggest that the basic supranucleosomal structure of chromatin is a zigzag helical ribbon with a repeat unit made of two nucleosomes connected by a relaxed spacer DNA. A remarkable feature of one particular ribbon is that it solves the apparent paradox between the number of DNA turns per nucleosome and the total linking number of a nucleosome-containing closed circular DNA molecule. We show here that the repeat unit of the proposed structure, which contains two nucleosomes with -13/4 DNA turns per nucleosome and one spacer crossover per repeat, contributes -2 to the linking number of closed circular DNA. Space-filling models show that the cylindrical 250-A chromatin fiber can be generated by twisting the ribbon.It is now well-established that the unit structure of chromatin, the nucleosome (1-3), is a flat cylindrical particle (110 X 110 x 57 A) with DNA wrapped around a histone octamer in a lefthanded toroidal supercoil of approximately 80 base pairs (bp) per turn (4). There are 146 bp of DNA in the nucleosome core describing -13/4 superhelical turns (5) and 20-95 bp of spacer DNA between neighboring nucleosome cores (6).The nature of the supranucleosomal structure of chromatin is less clear. Electron microscopic studies of eukaryotic nuclei have revealed a "thin" 100-A chromatin fiber and a "thick" 250-A fiber (7-9). Many past models have assumed that neighboring nucleosomes are stacked side by side to generate a 100-A nucleofilament, which is further coiled into a 250-A "solenoid" (10, 11). The unstacked 100-A fiber ("beads on a string") can be readily observed in nuclei and chromatin preparations spread under isotonic conditions (1, 2, 12). A stacked 100-A nucleofilament also has been detected, but only at high salt concentrations (11). Although the 100-A fiber is present in histone Hi-depleted chromatin, the 250-A fiber is never observed under these conditions (11, 12). Thus, histone HI must play a role in the further compaction of the linear chain of beads (12, 13). Reconstitution studies with the four intranucleosomal histones and small circular DNAs in the presence of nicking-closing enzyme have revealed that the DNA coiling around a nucleosome changes the DNA linking number by -1 (14). The histone Hl-induced compaction of the "beads on a string" into a 250-A-diameter supranucleosomal structure (15) does not cause further changes in the linking number (16). Because the nicking-closing enzyme relaxes the spacer DNA (16), the observed change in the linking number must be due to the particular DNA structure in the nucleosome.The linking number L of a closed circular DNA (ccDNA) molecule is the number of topological revolutions made by one strand about the other [counted after the molecule is constrained to lie in a plane (17)]. L is a topological invariant (i.e., an integer which is unchanged by all deformations that leave the strands intact). Moreover, it is related to the writhing number W and the twist number T by the equation (18-21)W is a real number that measures the shape...