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Cleavage of pBR322 DNA I by the restriction endonuclease HinfI is preferentially inhibited at specific HinfI cleavage sites. These sites in pBR322 DNA I have been identified and ordered with respect to the frequency with which they are cleaved. The HinfI site most resistant to cleavage in pBR322 DNA I is unique in that runs of G-C base pairs are immediately adjacent on both sites. Two differently permuted linear (DNA III) species were produced by cleavage with two different restriction endonucleases, PstI and AvaI. Only one of these linear molecules, that produced by PstI, exhibits the same preferential cleavage pattern as DNA I. The second linear species, that arising from AvaI digestion, shows pronounced relative inhibition of cleavage at the HinfI sites nearest the ends of the molecule (100 to 120 base pairs away, respectively). This result suggest that proximity to the termini of a linear DNA molecule might also influence preferential cleavage. The possibility of formation of stem-loop structures does not appear to influence preferential cleavage by HinfI.

The geometric properties of duplex DNA are systematically altered when the DNA is wrapped on a protein surface. The linking number of surface-wrapped closed circular DNA is the sum of two integers: the winding number, phi, a function of the helical repeat; and the surface linking number, SLk, a newly defined geometric constant that accounts for the effects of surface geometry on the twist and writhe of DNA. Changes in the helical repeat, h, and in the winding number can be deduced solely from surface geometry and superhelix density, sigma. This treatment relates the theoretically important properties twist and writhe to the more experimentally accessible quantities phi, h, SLk, and sigma. The analysis is applied to three biologically important cases: interwinding of DNA in a plectonemic superhelix, catenated DNA, and minichromosomes.

The presence of certain local structural elements in superhelical DNA, such as cruciforms and denatured loops, complicates the topological and geometric analysis of these molecules. In particular, the duplex axis is often difficult to define. In consequence, the usual conservation condition, Lk = Tw + Wr, is often inapplicable as formulated in terms of the winding of either strand of the DNA about the duplex axis. We present here a more general formulation of the topological conservation condition in terms of a model in which the two strands of DNA are regarded as twisting about one another, and in which one of the two strands is considered to writhe. We define a ladder-like correspondence surface, which connects the two strands nd is independent of whether or not a unique duplex axis is locally available. These considerations lead to the definition of a new topological property of superhelical DNA, the intersection number, In. This quantity describes the complexity of a local structural element; in the case of a cruciform, for example, the intersection number is a measure of the number of duplex turns removed from the major segment of the DNA by the cruciform formation. In more general terms, the topological constraint applicable to closed circular DNA is given by Lk(W,C) + In(S,C) = Tw(W,C) + Wr (C), where W and C represent the two strands of the DNA and S is the ladder-like correspondence surface that connects the two strands.

The complexes 1,10-phenanthrolineethylenediamineplatinum(II) and 2,2'-bipyridineethylenediamineplatinum(II) have a planar, aromatic ligand system that facilitates intercalation, as shown by their ability to unwind closed circular duplex DNA. Nonbonded steric interactions can rotate the pryidine ligands out of the coordination plane in bis(pyridine)ethylenediamineplatinum(II), thus preventing intercalation. Fiber x-ray diffraction patterns of the two metallointeracalators indicate that the binding is governed by the neighbor exclusion principle.

The time course of appearance of type I topoisomerase activity after the infection of mouse L cytoplasts by vaccinia virus was determined. When the enucleation procedure was carried out with unsynchronized cell cultures, a high level of host-cell-specific type I topoisomerase activity was found associated with the resulting cytoplasts. If cells were first synchronized by the two-cycle thymidine block method and then enucleated after release, the level of host type I topoisomerase activity was also high for S-phase-enucleated cells but was very low for cytoplasts prepared from cells previously synchronized and enucleated during either the G1 or the G2 phase. After the infection of G1-phase-enucleated cytoplasts with vaccinia virus, newly synthesized type I topoisomerase activity first appeared at about 3 h postinfection. Virosomes were isolated from the infected, synchronized cytoplasts and assayed for the presence of type I topoisomerase activity. The activity remained at the top of a sucrose gradient, well resolved from the virosome fraction, at the low salt levels (0.01 M KCl, 0.01 M Tris hydrochloride, pH 8.0) normally used in the course of virosome purification. If the sedimentation was at the higher salt concentration (0.15 M KCl) at which the enzyme shows optimal activity, type I topoisomerase cosedimented with the virosome fraction onto the sucrose gradient cushion. These results show that the type I topoisomerase activity dependent upon vaccinia virus infection may be detected with high sensitivity in G1-phase-enucleated cytoplasts. The association with virosomes is consistent with an involvement of topoisomerase activity either in DNA replication or in late transcription.

The melting transition for closed, underwound DNAs and for nicked or linear DNAs was monitored by velocity sedimentation and by absorbance spectroscopy in aqueous NaCCl3CO2 (NaTCA) and RbTCA. The addition of neutral trichloroacetate lowers the midpoint of the helix-coil transition by 26% C/M for RbTCA and by 32% C/M for NaTCA, depressing the denaturation region to near room temperature at neutral pH. The melting of nicked DNA is cooperative, occurring over a temperature range of about 5.6 degrees C. The melting profile for closed DNA is broad and noncooperative with a transition breadth greater than 45 degrees. Closed DNAs undergo a structural alteration, as revealed by velocity sedimentation, resulting in a reduction in the number of superhelical turns at temperatures and salt concentrations substantially below the melting temperatures and salt concentrations substantially below the melting temperature of the nicked DNA. The reduction in the extent of supercoiling continues upon isothermal addition of salt up to the salt concentration at which all superhelical turns are removed. The salt concentration at the principal minimum in the sedimentation velocity profile (3.16 M NaTCA for PM-2 DNA) is approximately the same as that at the midpoint of the helix-coil transition for the nicked DNA.

Aqueous RbTCA permits the buoyant banding of both native and denatured DNA at room temperature and neutral pH. A unique property of this solvent is the bouyant resolution of closed circular, underwound DNA (I) from the corresponding nicked (II) species. Conditions are reported here in which PM-2 DNA I is physically resolved from native PM-2 DNA II, the buoyant separation being 1.27 mq/ml in 3.3 M RbTCA at 25 degrees C. The separation between nicked and closed DNAs increases with temperature up to 35.5 degrees C, at which PM-2 DNA II cooperatively melts and subsequently pellets. The isothermal buoyant density of a cloed DNA increases linearly as the linking number (Lk) of the closed DNA decreases. The early melting of closed DNA may be monitored with high precision by buoyant banding in RbTCA, it being possible to detect the disruption of as few as 40 base pairs in PM-2 DNA (10,000 base pairs). The constraint that the linking number be conserved in closed DNA requires that a change in duplex winding be accompanied by a compensating change in supercoiling. We estimate the linking number deficiency of PM-2 DNA I to be 0.094 turns per decibase pair. This result permits the estimation of the EtdBr unwinding angle, phi, by comparison with alternative determinations of the linking number deficiency which depend upom the value of phi. The result obtained here is that phi = 27.7 degrees +/- 0.5 degrees and is approximately independent of temperature over the range 15 degrees-35 degrees.

The four identical recognition sites for the restriction endonuclease PstI in purified plasmid pSM1 DNA I are cleaved at markedly different rates. The order and relative frequencies of cleavage at these four PstI sites have been determined from the order of appearance of partial cleavage products and from an analysis of production of specific unit length linear molecules. The same pattern of preferential cleavage is also found when linear, nicked circular, or relaxed closed circular forms of the same plasmid DNa are used as substrates for PstI. Inspection of the nucleotide sequences immediately adjoining each of the PstI sites suggests that the presence of adjacent runs of G-C base pairs confers significant resistance to cleavage.

The melting transition of the magnesium salt of DNA has been systematically examined in the presence of various types of anions. The addition of ClO4- to a concentration of 3.0 N results in the biphasic optical transition, with the first phase exhibiting rapid reversibility and independence of the DNA concentration. This subtransition, which is interpreted as an intramolecular condensation to a collapsed form of DNA, is followed by a DNA concentration-dependent aggregation reaction. The aggregation can be reversed by increasing the ClO4- concentration to 6.0 N while elevating the temperature to post-transition levels. Alternatively, both the collapse and the aggregation can be prevented by melting in the presence of trichloroacetate, the most strongly chaotropic solvent for DNA which has been reported (K. Hamaguchi and E. P. Geiduschek (1962), J. Am. Chem. Soc. 84, 1329). The forces responsible for mediating both the collapse and the aggregation are superficially similar to those involved in maintaining duplex stability. The collapsed form, in particular, possibly possesses features in common with the condensed structures which can be produced in aqueous solution of certain polymers, such as polyethylene glycol (Lerman, L.S. (1971), Proc. Natl. Acad. Sci. U.S.A. 68, 1886).

The interaction of platinum and palladium complexes with closed and nicked circular and linear DNAs was investigated by a variety of methods. Cationic metal complexes containing flat, aromatic ligands, such as 2,2',2''-terpyridine, o-phenanthroline, and 2,2'-bipyridine, interfere with the usual fluorescence enhancement of ethidium bromide by competing for intercalation sites on calf-thymus DNA. Metal complexes having kinetically exchangable ligands, including the antitumor drugs cis-[(NH3)2PtCl2] and [(en)PtC12], inhibit noncompetitively the DNA-associated ethidium fluorescence enhancement by binding covalently to the bases and blocking potential intercalation sites. Only the metallointercalators were capable of altering the DNA duplex winding, as judged by the effects of these reagents upon the electrophoretic mobility and sedimentation behavior of PM-2 DNAs. Long-term (t greater than 120 h) interactions of metal complexes with PM-2 DNAs I, I0, and II, corresponding to superhelical, closed relaxed, and nicked circles, respectively, showed that covalent binding occurs the most readily to DNA I, possibly because of the presence of underwound duplex regions in this tightly wound superhelical DNA. The active antitumor drugs cis-[(NH3)2PtC12] and [(en)PtC12] bind covalently to DNA I under conditions where the inactive trans- [(NH3)2PtC12] does not. Most of the complexes studied were capable of producing chain scissions in PM-2DNA I. Exceptions are the kinetically inert complexes [(bipy)Pt(en)]2+ and (terpy)Pt(SCH2CH2OH)]+, suggesting that covalent binding might be a prerequisite for nicking.

The wrapping of closed circular DNA on a protein surface, followed by relaxation with a topoisomerase and removal of proteins, produces a characteristic DNA linking deficiency, delta Lk. We show that the magnitude of delta Lk depends upon the surface shape, and we calculate changes in delta Lk caused by particular distortions of the protein wrapping surface. If the DNA remains attached to the surface during distortion, the DNA winding number, phi, is not altered. The change in delta Lk is then equal to the change in the surface linking number, SLk, which is a straightforward measure of the wrapping of the DNA around the surface. For left-handed wrapping, as in a nucleosome, SLk = -n, the number of times that the DNA axis winds around the axis of the protein complex. We calculate values of SLk for the helical wrapping of a constant length of DNA on protein surfaces having the shapes of cylinders and of ellipsoids and hyperboloids of revolution. If the equatorial radius of the protein is fixed, change in shape from a cylinder to a hyperboloid increases SLk, while the corresponding change to an ellipsoid reduces SLk. We apply the general results to the interpretation of experiments in which minichromosomes are relaxed with topoisomerase at various temperatures and delta Lk is determined. The result is that a distortion of the nucleosome core by at most 5% (the change in the radius at the axial extremity relative to the equator) is sufficient to explain the observed delta Lk changes.

Multiple DNA-dependent enzyme activities have been detected in highly purified preparations of a single-strand-specific nuclease from vaccinia virus. These enzyme preparations were extensively purified and characterized by using superhelical DNAs as substrates. In particular, the nuclease activity was monitored by the extent of conversion of supercoiled closed duplex DNA (DNA I) to nicked circular DNA (DNA II), which could subsequently be converted to duplex linear DNA (DNA III) by prolonged incubation with the enzyme. DNA species which were not substrates for the enzyme included relaxed closed duplex DNA, DNA II which had been prepared by nuclease S1 treatment or by photochemical nicking of DNA I, and DNA III. With plasmid pSM1 DNA as substrate, the extent of cleavage of DNA I to DNA II was found to increase with superhelix density above a threshold value of about -0.06. The linear reaction products were examined by gel electrophoresis after restriction enzyme digestion of the DNAs from plasmids pSM1 and pBR322 and of the viral DNAs from bacteriophage phi X174 (replicative form) and simian virus 40, and the map coordinate locations of the scissions were determined. These products were further examined by electron microscopy and by gel electrophoresis under denaturing conditions. Electron micrographs taken under partially denaturing conditions revealed molecules with terminal loops or hairpins such as would result from the introduction of cross-links at the cutting sites. These species exhibited snapback renaturation. The denaturing gel electrophoresis experiments revealed the appearance of new bands at locations consistent with terminal cross-linking. With pSM1 and pBR322 DNAs, this band was shown to contain DNA that was approximately twice the length of a linear single strand. The terminal regions of the cross-linked linear duplex reaction products were sensitive to nuclease S1 but insensitive to proteinase K, suggesting that the structure is a hairpin loop not maintained by a protein linker. A similar structure is found in mature vaccinia virus DNA.

One of the major DNA binding proteins contained in vaccinia virus is an 11-kDA species denoted VP11. The biosynthesis of VP11, a late polypeptide, occurs subsequent to the initiation of viral DNA replication. In particular, VP11 synthesis is blocked by cytosine arabinoside, a specific inhibitor of DNA synthesis. We show here that VP11 is specifically phosphorylated subsequent to translation. Phosphorylated VP11 is present both in viral core particles and in the cytoplasm of virus-infected cells. Kinetic analysis reveals that the total amount of phosphorylated VP11 species increases rapidly and remains approximately constant for as long as 17 hr postinfection. Phosphorylation occurs at two different serine residues, progressing from either site singly to the diphosphorylated product. Under steady-state conditions, the phosphorylated derivative constitutes approximately 85% of total VP11 in extracts of vaccinia virus-infected cells. Even though 15% of the VP11 remains unphosphorylated in cell extracts, only phosphorylated VP11 is found in mature viral cores.

The vaccinia virus nicking-joining (NJ) enzyme has been purified to homogeneity from a preparation of virus cores. The virus-specific DNA-dependent enzyme, which does not require ATP, is a single polypeptide of Mr 50,000 and possesses both endonuclease and ligase activities. The principal end product of the enzyme activity, following incubation with closed circular DNA of sufficient linking deficiency, is a linear DNA in which one of the termini has become cross-linked by the in vitro formation of a hairpin. The ability of the NJ enzyme to cross-link DNA is significantly enhanced by in vitro proteolysis. The enzymatic properties of the proteolytic digestion product, a 44-kDa polypeptide, differ in several other ways from the intact NJ enzyme. In particular, the specific activity is enhanced and the ionic strength optimum is shifted toward higher salt concentrations. It is suggested that the purified 50-kDa species is a pronuclease that is activated by proteolytic processing.

The antitumor drug cis-dichlorodiammineplatinum(II) (cis-DDP) and the inactive trans isomer bind and produce cooperative changes in closed and nicked circular duplex DNA's. Covalent binding of both platinum complexes to the closed circular DNA alters the degree of supercoiling, presumably by disrupting and unwinding the double helix. Electron micrographs show the platinated DNA's to be shortened by up to 50 percent of their original length. At similar ratios of bound platinum per nucleotide, the electrophoretic mobilities of the DNA's in gels containing the dye ethidium bromide are the same for both isomers. The only detectable difference in the binding of the two platinum isomers is an increase in the electrophoretic mobility in nondye gels of closed circular DNA having small amounts of bound cis-DDP that is not apparent for the trans complex.