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![]() ![]() Hence, we asked whether other DNA molecules, of length and sequence different than P4 DNA, could be packaged inside P4 capsids and recovered also as highly knotted forms. However, one drawback of this natural system is that knot formation is restricted to the viral DNA. Otherwise the knots can be readily converted into covalently closed DNA circles by a brief reaction with DNA ligase. Because DNA circularization results from non-covalent joining of 19-bp cohesive ends, the knots can be directly analyzed without requiring enzymatic nicking. ![]() P4 DNA knots are complex, heterogeneous and can be purified in preparative amounts. ![]() Subsequent research indicated that DNA knot formation was caused by the premature circularization of the P4 genome inside the small volume of the phage capsid. Formation of these knots was found enhanced in P4 phage derivatives with genome deletions and in tailless-mutants. These studies soon conducted to the discovery that a large fraction of DNA molecules extracted from phage P4 were highly knotted nicked DNA circles. Newly made P4 phages lead then to bacterial lysis and start a new infective cycle. Each P4 DNA circle is cleaved at the 55-bp cos sequence to produce a linear molecule with the 19-bp cohesive ends, which is actively threaded into a P4 capsid. Contrary to phage lambda, which packages its genome from replicated linear DNA multimers, P4 uses covalently closed DNA circles as a preferred substrate for DNA packaging. P4 uses then the machinery of P2 to amplify and package its own genome. When P4 infects a bacterial host lysogenic for P2, the 11.2 kb P4 genome is injected as a linear double-stranded DNA that quickly circularizes by ligation of its terminal cohesive ends. P4 is a satellite phage that needs the helper prophage P2 for proliferation. The mechanisms of genome propagation by phage P4 had been elucidated thanks to the studies of Richard Calendar and colleagues. Here we developed the P4 phage system as a convenient source of knotted DNA. To facilitate these studies, a method to produce abundant DNA knots would be highly valuable. Knotted DNA molecules will be also useful to further investigate biophysical properties of constrained DNA, as well as the activity of topoisomerases, recombinases and other DNA interacting ensembles. DNA knots could play a role in the high order organization of chromosomes, yet they should not interfere DNA replication and transcription. However, the biological relevance and potential applications of knotted DNA molecules remain to be explored. Also, the specific knot types produced when DNA recombinases and topoisomerases act on circular DNA was informative to reconstruct the architecture of these protein-DNA ensembles. Difference between plasmid and cosmid free#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. Knot analyses with these techniques had been very useful to infer physical properties of double stranded DNA. Along these findings, the development of electron microscopy for RecA-coated DNA molecules and of high resolution agarose gel electrophoresis allowed the identification of numerous knot types shaped into DNA. Later on, DNA knots were discovered in plasmids undergoing transcription and replication in bacteria with deficient topoisomerase activity. DNA knots were first observed in vitro near four decades ago in single-stranded DNA rings incubated with bacterial topoisomerase I and in double-stranded DNA molecules extracted from phage P4. The occurrence of knotted DNA molecules is common in biological systems. These molecules can be produced in preparative amounts suitable for systematic studies and applications. With this biological system, DNA molecules of varying length and sequence can be shaped into very complex and heterogeneous knotted forms. Over 250 μg of knotted cosmid were typically obtained from 1 liter of bacterial culture. Such cosmids were then recovered as a complex mixture of highly knotted DNA circles. We constructed DNA cosmids as small as 5 kb and introduced them inside P4 capsids. We demonstrate here that this mechanism is not restricted to the viral genome. 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. A convenient method to produce abundant highly knotted DNA would be highly valuable for these studies. Moreover, potential applications of DNA molecules condensed by means of knotting remain to be explored. Yet, functional implications of knotted DNA are not fully understood. The formation of DNA knots is common during biological transactions. ![]() |
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