Knotted DNA from bacteriophage capsids.

Abstract

The majority of the DNA prepared from tailless capsids of bacteriophage P2 by the phenol extraction procedure consists of monomeric rings that have their cohesive ends joined. Electron microscopic and ultracentrifugal studies indicate that these molecules have a complex structure that is topologically knotted; they have a more compact appearance and a higher sedimentation coefficient when compared with regular nicked P2 DNA rings. Linearization of these rings by thermal dissociation or repair of the cohesive ends by DNA polymerase I in the presence ofall four deoxynucleoside triphosphates gives molecules that are indistinguishable from normal P2 DNA that has been similarly treated. The knotted nature of the majority of P2 head DNA is further supported by analyzing the products when these molecules are treated with ligase and the ligase-treated molecules are subsequently nicked randomly with DNase I. The data are consistent with the notion that, if such a molecule is first converted to a form that contains only one single-chain scission per molecule, strand separation gives a linear strand and a highly knotted singlestranded ring. The results suggest that the DNA packaged in tailless P2 capsids is arranged in a way that leads to the formation of a complex knot when the ends join. In an intact phage particle, the anchoring of one terminus of the DNA to the head-proximal end of the tail [Chattoraj, D. K. & Inman, R. B. (1974) J. MoL BioL 87, 11-22] presumably diminishes or prevents this kind ofjoining. The novel knotted DNA can be used to assay type II DNA topoisomerases that break and rejoin DNA in a double-stranded fashion.Mature P2 phage DNA consists of double-stranded molecules 33 kilobases long (1, 2) with 19-base cohesive ends (3, 4) that allow the formation of circular and concatemeric molecules in solution (1, 2). P2 phage preparations typically contain equal proportions of viable phage particles and tailless DNA-containing capsids (heads) (5). The two cohesive ends of the P2 DNA in the mature phage are not hydrogen bonded and are asymmetrically oriented with the left end anchored on the headproximal end ofthe tail (6). When DNA is extracted from a concentrated preparation of mature P2 phage, a viscous solution is obtained, due to the formation of hydrogen-bonded concatemers via pairing of the cohesive ends (1). We observed, however, that DNA extracted from concentrated P2 heads is much less viscous than the corresponding DNA from mature phage particles. In this communication, we present evidence that the DNA from tailless capsids (henceforth referred to as head DNA) represents a novel class of topological isomers of hydrogenbonded monomeric circular P2 DNA. The apparent low viscosity ofhead DNA relative to that of DNA from mature phages (henceforth referred to as phage DNA) is due to the presence of complex topological knots. MATERIALS AND METHODSBacteria and Bacteriophages. Escherichia coli C strains Cla (7) and C-520 (8), carrying the amber suppressor supD, were used as hosts to grow stocks of p...

Topological friction strongly affects viral DNA ejection

“…Increasing evidence shows that this cholesteric interaction is not only important for the formation of cholesteric phases in concentrated solutions of DNA (5-8) but can favor the spool-like DNA arrangements of viral DNA (9-11) inside small capsids. Moreover it can control the complexity of DNA self-entanglement in the form of knots (10,(12)(13)(14). It is useful here to recall that DNA knots have been already reported for some bacteriophages (12,15,16), although it is not yet clear how virus-specific effects (such as the genome anchoring to the capsid) may affect knot type and abundance.…”

Knotting probability of DNA molecules confined in restricted volumes: DNA knotting in phage capsids

“…Significant numbers of DNA knots are found also in biological systems: in Escherichia coli cells harboring mutations in the GyrB or GyrA genes (9), bacteriophages P2 and P4 (10,11), and cauliflower mosaic viruses (12). However, very little information about these systems has been inferred from the observed knots.…”

Sedimentation of knotted polymers

“…DNA knots are even more common within bacteriophage heads [15,16]. Arsuaga et al showed that the tight geometric confinement the genome is subjected to is a significant contributor to the knot formation [7,17].…”