Production of highly knotted DNA by means of cosmid circularization inside phage capsids

We had previously shown that circularization of the 11.2 kb linear DNA of phage P4 inside its viral capsid generates complex knots by the effect of confinement. We demonstrate here that this mechanism is not restricted to the viral genome. We constructed DNA cosmids as small as 5 kb and introduced them inside P4 capsids. Such cosmids were then recovered as a complex mixture of highly knotted DNA circles. Over 250 μg of knotted cosmid were typically obtained from 1 liter of bacterial culture.With this biological system, DNA molecules of varying length and sequence can be shaped into very complex and heterogeneous knotted forms. These molecules can be produced in preparative amounts suitable for systematic studies and applications.The occurrence of knotted DNA molecules is common in biological systems. DNA knots were first observed in vitro near four decades ago in single-stranded DNA rings incubated with bacterial topoisomerase I [1] and in double-stranded DNA molecules extracted from phage P4 [2,3]. Later on, DNA knots were discovered in plasmids undergoing transcription and replication in bacteria with deficient topoisomerase activity [4,5]. Along these findings, the development of electron microscopy for RecA-coated DNA molecules [6,7] and of high resolution agarose gel electrophoresis [8-11] allowed the identification of numerous knot types shaped into DNA. Knot analyses with these techniques had been very useful to infer physical properties of double stranded DNA. For instance, the effective diameter of duplex DNA was determined from the fraction of knotted circles found after the circularization of linear DNA molecules that joined cohesive ends in free solution [12,13]. Also, the specific knot types produced when DNA recombinases [14,15] and topoisomerases [16,17] act on circular DNA was informative to reconstruct the architecture of these protein-DNA ensembles. However, the biological relevance and potential applications of knotted DNA molecules remain to be e