When linear double-stranded DNA is packed inside bacteriophage capsids, it becomes highly compacted. However, the phage is believed to be fully effective only if the DNA is not entangled. Nevertheless, when DNA is extracted from a tailless mutant of the P4 phage, DNA is found to be cyclic and knotted (probability of 0.95). The knot spectrum is very complex, and most of the knots have a large number of crossings. We quantified the frequency and crossing numbers of these knots and concluded that, for the P4 tailless mutant, at least half the knotted molecules are formed while the DNA is still inside the viral capsid rather than during extraction. To analyze the origin of the knots formed inside the capsid, we compared our experimental results to Monte Carlo simulations of random knotting of equilateral polygons in confined volumes. These simulations showed that confinement of closed chains to tightly restricted volumes results in high knotting probabilities and the formation of knots with large crossing numbers. We conclude that the formation of the knots inside the viral capsid is driven mainly by the effects of confinement.

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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...