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Topoisomerase IV (Topo IV), an essential ATP-dependent bacterial type II topoisomerase. transports one segment of DNA through a transient double-strand break in a second segment of DNA. In vivo, Topo IV unlinks catenated chromosomes before cell division and relaxes positive supercoils generated during DNA replication. In vitro, Topo IV relaxes positive supercoils at least 20-fold faster than negative supercoils. The mechanisms underlying this chiral discrimination by Topo IV and other type II topoisomerases remain speculative. We used magnetic tweezers to measure the relaxation rates of single and multiple DNA crossings by Topo IV. These measurements allowed us to determine unambiguously the relative importance of DNA crossing geometry and enzymatic processivity in chiral discrimination by Topo IV. Our results indicate that Topo IV binds and passes DNA strands juxtaposed in a nearly perpendicular orientation and that relaxation of negative supercoiled DNA is perfectly distributive. Together, these results suggest that chiral discrimination arises primarily from dramatic differences in the processivity of relaxing positive and negative supercoiled DNA: Topo IV is highly processive on positively supercoiled DNA, whereas it is perfectly distributive on negatively supercoiled DNA. These results provide fresh insight into topoisomerase mechanisms and lead to a model that reconciles contradictory aspects of previous findings while providing a framework to interpret future results. [ABSTRACT FROM AUTHOR]

Phase-locking (frequency entrainment) of an oscillator, in which a periodic extrinsic signal drives oscillations at a frequency different from the unperturbed frequency, is a useful property for study of oscillator stability and structure. The cell cycle is frequently described as a biochemical oscillator; however, because this oscillator is tied to key biological events such as DNA replication and segregation, and to cell growth (cell mass increase); it is unclear whether phase locking is possible for the cell cycle oscillator. We found that forced periodic expression of the G[sub1] cyclin CLN2 phase locks the cell cycle of budding yeast over a range of extrinsic periods in an exponentially growing monolayer culture. We characterize the behavior of cells in a pedigree using a return map to determine the efficiency of entrainment to the externally controlled pulse. We quantify differences between mothers and daughters and how synchronization of an expanding population differs from synchronization of a single oscillator. Mothers only lock intermittently whereas daughters lock completely and in a different period range than mothers. We can explain quantitative features of phase locking in both cell types with an analytically solvable model based on cell size control and how mass is partitioned between mother and daughter cells. A key prediction of this model is that size control can occur not only in G1, but also later in the cell cycle under the appropriate conditions; this prediction is confirmed in our experimental data. Our results provide quantitative insight into how cell size is integrated with the cell cycle oscillator. [ABSTRACT FROM AUTHOR]

The recent development of new techniques to manipulate single DNA molecules has opened new opportunities for the study of the enzymes that control DNA topology: the type I and II topoisomerases. These single-molecule assays provide a unique way to study the uncoiling of single supercoiled DNA molecules and the unlinking of two intertwined DNAs. They allow for a detailed characterization of the activity of topoisomerases, including the processivity, the chiral discrimination, and the dependence of their enzymatic rate on ATP concentration, degree of supercoiling, and the tension in the molecule. These results shed new light on the mechanism of these enzymes and their function in vivo. [ABSTRACT FROM AUTHOR]

Over the past 10 years a number of new techniques have emerged that allow the manipulation of single DNA molecules and other biopolymers (RNA, proteins, etc.). These experiments have permitted the measurement of the DNA stretching and twisting elasticity and have consequently revealed the essential role played by the DNA mechanical properties in its interactions with proteins. We shall first describe the different methods used to stretch and twist single DNA molecules. We will then focus on its behaviour under torsion, especially by discussing the different methods used to estimate its torsional modulus. [ABSTRACT FROM AUTHOR]

Type-II topoisomerases are responsible for untangling DNA during replication by removing supercoiled and interlinked DNA structures. Using a single-molecule micromanipulation setup, we follow the real-time decatenation of two mechanically braided DNA molecules by Drosophila melanogaster topoisomerase (Topo) II and Escherichia coli Topo IV. Although Topo II relaxes left-handed (L) and right-handed (R-) braids similarly at a rate of ≈2.9 s[sup -1], Topo IV has a marked preference for L-braids, which it relaxes completely and processively at a rate of ≈2.4 s[sup -1]. However, Topo IV can unlink R-braids at about half that rate when they supercoil to form L-plectonemes. These results imply that the preferred substrate for unlinking by Topo IV has the symmetry of an L-crossing and shed new light on the decatenation of daughter strands during DNA replication, which are usually assumed to be linked in an R-braid. [ABSTRACT FROM AUTHOR]

xis 5 of the 2009-2013 Cancer Plan: 'Living with the disease and after it.' This 'provocative' heading emphasises a metaphor of life and of the passage of time in association with the word cancer, which more often carries images of cessation, termination and death. [ABSTRACT FROM AUTHOR]