DNA knots and DNA supercoiling.

Type II DNA topoisomerases permit passages of double stranded DNA segments through each other and this is achieved via a complex mechanism involving a transient cleavage of one duplex, a passage of the second duplex through the topoisomerase-spanned cleavage site and finally resealing of the cut duplex. Type II DNA topoisomerases facilitate many DNA transactions requiring manipulation of long DNA molecules and are necessary for the completion of such vital processes as DNA replication. Type II DNA topoisomerases are, however, doubly edged swords: whereas most of the catalysed passages are beneficial, some can be deleterious to living cells. When passages lead to formation of knots these impede transcription and replication and need to be eliminated quickly. Elimination of knots cannot be simply done by random passages since in long DNA molecules crammed within a small volume, such as a bacterial nucleoid, random passages would only result in formation of highly complicated Gordian knots. In 1997, Rybenkov et al. provided a partial explanation to the DNA knotting problem by demonstrating that type IIA DNA topoisomerases acting in vitro on relaxed DNA plasmids maintain the knotting level up to 50-fold below the level that would be obtained after random passages. However, the same reactions also demonstrated that this unknotting ability of type IIA DNA topoisomerases sharply decreases with DNA length. Since then, two puzzling questions were disputed by the topoisomerase community: (1) How can type IIA DNA topoisomerases preferentially accomplish intersegmental passages leading to unknotting while avoiding DNA knots and DNA supercoiling