Icosahedral bacteriophages pack their double-stranded DNA genomes to near-crystalline density and achieve one of the highest levels of DNA condensation found in nature. Despite numerous studies, some essential properties of the packaging geometry of the DNA inside the phage capsid are still unknown. We present a different approach to the problems of randomness and chirality of the packed DNA. We recently showed that most DNA molecules extracted from bacteriophage P4 are highly knotted because of the cyclization of the linear DNA molecule confined in the phage capsid. Here, we show that these knots provide information about the global arrangement of the DNA inside the capsid. First, we analyze the distribution of the viral DNA knots by high-resolution gel electrophoresis. Next, we perform Monte Carlo computer simulations of random knotting for freely jointed polygons confined to spherical volumes. Comparison of the knot distributions obtained by both techniques produces a topological proof of nonrandom packaging of the viral DNA. Moreover, our simulations show that the scarcity of the achiral knot 41 and the predominance of the torus knot 51 over the twist knot 52 observed in the viral distribution of DNA knots cannot be obtained by confinement alone but must include writhe bias in the conformation sampling. These results indicate that the packaging geometry of the DNA inside the viral capsid is writhe-directed.bacteriophage ͉ DNA condensation ͉ DNA electrophoresis ͉ Monte Carlo simulation ͉ DNA writhe A ll icosahedral bacteriophages with double-stranded DNA genomes are believed to pack their chromosomes in a similar manner (1). During phage morphogenesis, a procapsid is first assembled, and a linear DNA molecule is actively introduced inside it by the connector complex (2, 3). At the end of this process, the DNA and its associated water molecules fill the entire capsid volume, where DNA reaches concentrations of 800 mg͞ml (4). Some animal viruses (5) and lipo-DNA complexes used in gene therapy (6) are postulated to hold similar DNA arrangements as those found in bacteriophages.Although numerous studies have investigated the DNA packing geometry inside phage capsids, some of its properties remain unknown. Biochemical and structural analyses have revealed that DNA is kept in its B form (7-9) and that there are no specific DNA-protein interactions (10, 11) or correlation between DNA sequences and their spatial location inside the capsid, with the exception of the cos ends in some viruses (12). Many studies have found that regions of the packed DNA form domains of parallel fibers, which in some cases have different orientations, suggesting a certain degree of randomness (8,9,13,14). The above observations have led to the proposal of several long-range organization models for DNA inside phage capsids: the ball of string model (13), the coaxial spooling model (8,11,13,14), the spiral-fold model (15), and the folded toroidal model (16). Liquid crystalline models, which take into account properties of DNA at high concen...

) demonstrated that novel 6,8-difluoroquinolones were potent effectors of eukaryotic topoisomerase II. To determine the contribution of the C-8 fluorine to drug potency, we compared the effects of -(4-hydroxyphenyl)-1-cyclopropyl-4-quinolone-3-carboxylic acid] on the enzymatic activities of Drosophila melanogaster topoisomerase II with those of 953 (the 6,955). Removal of the C-8 fluoro group decreased the ability of the quinolone to enhance enzyme-mediated DNA cleavage -2.5-fold. Like its difluorinated counterpart, CP-115,955 increased the levels of cleavage intermediates without impairing the DNA religation reaction of the enzyme. Removal of the C-8 fluorine reduced the ability of the quinolone to inhibit topoisomerase II-catalyzed DNA relaxation. In addition, the cytotoxicity of CP-115,955 towards Chinese hamster ovary cells was decreased compared with that of CP-115,953. These results demonstrate that the C-8 fluorine increases the potency of quinolone derivatives against eukaryotic topoisomerase II and mammalian cells. Further comparisons of 804 (the N-1 ethyl-substituted derivative of the difluoro parent compound) indicate that the two intrinsic activities of quinolone-based drugs towards topoisomerase II (i.e., enhancement of DNA cleavage and inhibition of catalytic strand passage) can be differentially influenced by alteration of ring substituents. Finally, correlations between the biochemical and cytological activities of these drugs suggest that the ability to inhibit catalytic strand passage enhances the cytotoxic potential of quinolones towards eukaryotic cells.Topoisomerase II is an essential enzyme (9,21,23,53) that is required for chromosome structure (5,13,14,16,17), condensation (1, 36, 52, 56), and segregation (9,23,47,54). It also appears to play roles in DNA replication, transcription, and recombination in eukaryotic cells (3,6,8,30,39,43,47,51,55).In addition to its cellular functions, topoisomerase II is the primary target for several classes of antineoplastic drugs (32,48,59). These agents are widely used for the treatment of human cancers (32,48,59) and their clinical efficacies correlate with their abilities to stabilize covalent enzymecleaved DNA complexes that are intermediates in the catalytic cycle of the enzyme (31,32,43,48,59). Previous studies with etoposide (40, 46) and 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) (45, 46) demonstrated that these topoisomerase 1I-targeted drugs stabilize cleavage complexes primarily by inhibiting the ability of the enzyme to religate cleaved DNA.Recent work indicates that the DNA cleavage complex of eukaryotic topoisomerase II is also a target for novel 6,8-difluoroquinolone derivatives (4, 44). While quinolone-based drugs have been developed extensively as antimicrobial agents (targeted to DNA gyrase, the prokaryotic counterpart of topoisomerase II) (12,24,58), these studies provided evidence that quinolones may have potential as antineoplastic drugs. One of the difluoro compounds examined, 6,8-difluoro-7-(4-hydroxyphenyl)-1-cyclopro...

). However, the features of the drug that contribute to its activity towards mammalian systems have not been characterized. Therefore, CP-115,953 and a series of related quinolones were examined for their activity against calf thymus topoisomerase H and cultured mammalian cells. CP-115,953 stimulated DNA cleavage mediated by the type H enzyme with a potency that was -600-fold greater than that of the antimicrobial quinolone ciprofloxacin and -50-fold greater than that of the antineoplastic drug etoposide. As determined by the ability to enhance enzyme-mediated DNA cleavage, quinolone activity towards calf thymus topoisomerase H was enhanced by the presence of a cyclopropyl group at the N-1 ring position and by the presence of a fluorine at C-8. Furthermore, the 4'-hydroxyphenyl substituent at the C-7 position was critical for the potency of CP-115,953 towards the mammalian type H enzyme. In this regard, the aromatic nature of the C-7 ring as well as the presence and the position of the 4'-hydroxyl group contributed greatly to drug activity. Finally, the cytotoxicity of quinolones in the CP-115,953 series towards mammalian cells paralleled the in vitro stimulation of DNA cleavage by topoisomerase H rather than the inhibition of enzyme-catalyzed DNA relaxation. This correlation strongly suggests that these quinolones promote cell death by converting topoisomerase IT to a cellular poison.