Physical and Topological Properties of Circular DNA

252

320

The conformation and dynamics of circular polymers is a subject of considerable theoretical and experimental interest. DNA is an important example because it occurs naturally in different topological states, including linear, relaxed circular, and supercoiled circular forms. A fundamental question is how the diffusion coefficients of isolated polymers scale with molecular length and how they vary for different topologies. Here, diffusion coefficients D for relaxed circular, supercoiled, and linear DNA molecules of length L ranging from Ϸ6 to 290 kbp were measured by tracking the Brownian motion of single molecules. A topology-independent scaling law D ϳ L ؊ was observed with L ‫؍‬ 0.571 ؎ 0.014, C ‫؍‬ 0.589 ؎ 0.018, and S ‫؍‬ 0.571 ؎ 0.057 for linear, relaxed circular, and supercoiled DNA, respectively, in good agreement with the scaling exponent of Х 0.588 predicted by renormalization group theory for polymers with significant excluded volume interactions. Our findings thus provide evidence in support of several theories that predict an effective diameter of DNA much greater than the Debye screening length. In addition, the measured ratio DCircular͞ DLinear ‫؍‬ 1.32 ؎ 0.014 was closer to the value of 1.45 predicted by using renormalization group theory than the value of 1.18 predicted by classical Kirkwood hydrodynamic theory and agreed well with a value of 1.31 predicted when incorporating a recently proposed expression for the radius of gyration of circular polymers into the Zimm model. circular ͉ polymer ͉ polyelectrolyte ͉ hydrodynamics ͉ excluded volume W hile many eukaryotic genomes are linear, prokaryotic genomes and most cloned DNA constructs are circular (1). Indeed, a commonly stated motivation for theoretical calculations on circular polymers is that they may be applicable to understanding the behavior of DNA. However, four of the five previously reported studies on the diffusion of circular polymers have used synthetic polymers, and only two of these, both using synthetic polymers, examined the dependence of the diffusion coefficient on molecular length. The dependence of D on length for relaxed circular DNA has never been measured. Here, we examine linear, relaxed circular, and supercoiled DNA molecules covering a wide range of lengths (Ϸ6 to 290 kbp).For long linear polymers in a good solvent, where excluded volume effects are appreciable, polymer physics theory (2) predicts D ϳ 1͞R G ϳ L Ϫ with Х 0.588, where R G is radius of gyration. The same scaling exponent has been calculated for both dynamic (D ϳ L Ϫ ) and static (R G ϳ L ) quantities, with nearly identical results determined by using a wide range of methods. Static scaling has been examined by using Monte Carlo simulations (3), bead-rod simulations (4), and cylindrical selfavoiding polygon models (5). Renormalization group theory methods have been used in both static (3, 6) and dynamic (7) calculations, and bond-fluctuation simulations (8) were used to measure and compare both scaling relationships. The predicted scaling exponent is close ...

Section: Resultsmentioning

confidence: 98%

Diffusion of isolated DNA molecules: Dependence on length and topology

Robertson

Laib

Smith

2006

252

320

“…The first conclusion above is easy to understand because, if x is an integral multiple of the helical repeat, the twist number Tw and the linking number a will be increased by the same integral number, and therefore the writhing number Wr, which determines the electrophoretic mobility and is related to a and Tw by the equation a = Tw + Wr (11)(12)(13)(14), remains Fig. 1 bottom.…”

mentioning

confidence: 97%

Helical repeat of DNA in solution.

Wang¹

1979

449

193

The helical repeat of DNA in solution has been measured directly by analyzing the gel electrophoretic patterns of pairs of covalently closed DNAs with length differences between 1 and 58 base pairs, out of a total length of about 4350 base pairs per DNA molecule. The method is based on the observation that for a covalently closed DNA of a fixed size of n base pairs (n of the order of several thousand), under appropriate conditions, two topological isomers (topoisomers) differing by 1 in their linking numbers are well resolved by gel electrophoresis. If the size of the DNA is increased to n + x base pairs,

“…Such DNA molecules are subject to the topological constraint (11) (2,3,8,9), untwisting activity (5), relaxing activity (6,7), and nicking-closing activity (10 (14). For the preparation of labeled virus, HeLa S cells were suspended at 107/ml in 1:1 Puck's saline/Eagle's spinner medium (1/10 normal methionine concentration) and were infected with purified vaccinia virus at a multiplicity of infection of 4 The amount of protein was assessed by scintillation counting of incorporated [35S]methionine, based upon a specific radioactivity of 1.1 X 106 cpm/mg for whole virus.…”

mentioning

confidence: 99%

A DNA nicking-closing enzyme encapsidated in vaccinia virus: partial purification and properties.

Bauer¹,

Ressner²,

Kates³

et al. 1977

Vaccinia virus cores contain an activity which is able to relax both left-and right-handed superhelical DNA. This virus-specific nicking-closing enzyme has been highly purified and differs from the corresponding host enzyme in salt optimum, in sedimentation coefficient, and in polypeptide composition as determined on sodium dodecyl sulfate/polyacrylamide gels. The enzyme is probably newly synthesized after the cessation of host protein synthesis which follows virus infection. The most highly purified preparation contains two polypeptides, one of molecular weight 24,000 and the other 35,000. The former polypeptide is a major constituent of the virus (7% of total protein by weight), whereas the latter is present in a much smaller amount (0.2%). Chromatography with denatured DNA-cellulose reveals that the activity is predominately associated with those fractions enriched in the polypeptide of greater molecular weight. Many fundamental biological processes are associated with changes in the duplex winding of DNA. These include DNA replication; transcription; recombination; formation of nucleosomes; packaging of DNA in organelles, such as mitochondria or chloroplasts; formation of complex prokaryotic chromosomes (1); and encapsidation into viruses. Enzymes from several sources have recently been discovered which are able to relieve torsional stress on the DNA duplex. None of these appears to require a cofactor, and their primary function is to introduce a transient local swivel that allows the DNA to rotate in response to a torque.The prokaryotic enzymes, termed omega proteins (2), have been purified from Escherichia coli 1100 (2, 3) and from E. coli B (4) and are able to catalyze duplex rotation in the righthanded (overwinding) direction only. The eukaryotic enzymes are able to catalyze rotation in both directions and have so far been discovered in several animal cell nuclei (5-9). In the present report we show that such an activity is encapsidated in mature vaccinia virions and is active in viral cores, which are able to carry out transcription as intact complexes. The activity is not identical to the corresponding host protein and is newly synthesized following infection. This result, representing a virus-specific nicking-closing enzymet, confirms the widespread distribution of this activity.The only presently available assay for nicking-closing activity is based upon the ability of the enzyme to remove superhelical turns from closed circular duplex DNA. Such DNA molecules are subject to the topological constraint (11) that the net interstrand winding, a, is a constant and is the sum of the number MATERIALS AND METHODS Reagents. Yeast alcohol dehydrogenase and horse heart cytochrome c were obtained from Worthington and Sigma, respectively. Ethidium bromide (EtdBr) was obtained from Calbiochem, and [35S]methionine was purchased from New England Nuclear. All other chemicals were of reagent grade.Preparation of Virus. Vaccinia virus strain WR, grown in HeLa S cells, was purified by the method of Joklik (14). ...