Action of nicking-closing enzyme on supercoiled and nonsupercoiled closed circular DNA: formation of a Boltzmann distribution of topological isomers.

Pulleyblank, David E.; Shure, Mavis; Tang, David; Vinograd, Jerome; Vosberg, Hans−Peter

doi:10.1073/pnas.72.11.4280

Cited by 361 publications

(187 citation statements)

References 16 publications

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“…Variance of Writhe-Closed circular plasmid DNA contains different numbers of helical turns when the linear form with cohesive ends is circularized and ligated (11,12). This behavior is due to the fact that DNA is flexible and the helices bend and twist under the influence of thermal energy.…”

Section: Resultsmentioning

confidence: 99%

Triplet Repeat Instability and DNA Topology: An Expansion Model Based on Statistical Mechanics

Gellibolian

Bacolla

Wells

1997

Journal of Biological Chemistry

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The variance of writhe, the contribution of writhe to supercoiling, and the free energies of supercoiling were calculated for (CTG⅐CAG) n and (CGG⅐CCG) n triplet repeat sequences (TRS) by statistical mechanics from the bending and torsional moduli previously determined. Expansions of these sequences are inherited by nonmendelian transmission and are linked with several hereditary neuromuscular diseases. The variance of writhe was greater for the TRS than for random B-DNA. For random B-DNA, (CGG) n , and (CTG) n , the contribution of writhe to supercoiling was 70, 78, and 79%, whereas the free energy of supercoiling at a length of 10 kilobase pairs was 1040⅐RT, 760⅐RT, and 685⅐RT, respectively. These data indicate that the TRS are preferential sites for the partitioning of supercoiling. Calculations of the differences in free energy of supercoiling between the TRS and random B-DNA revealed a local minimum at ϳ520 base pairs. Human medical genetic studies have shown that individuals carrying up to 180 -200 copies of TRS (540 -600 base pairs, premutations) in the fragile X or myotonic dystrophy gene loci are usually asymptomatic, whereas large expansions (>200 repeats, full mutations), which lead to disease, are observed in their offspring. Therefore, the length corresponding to the local minimum in free energy of supercoiling correlates with the genetic breakpoint between premutation and full mutation. We propose that (a) TRS instability is mediated by DNA mispairing caused by the accumulation of supercoiling within the repeats, and (b) the expansions that take place at the premutation to full mutation threshold are associated with increased mispairing caused by the optimal partitioning of writhe within the TRS at this length.Several human loci associated with neurodegenerative disorders have been shown to carry a new form of mutation, i.e. the expansion of a DNA triplet repeat sequence (TRS) 1 with composition (CTG⅐CAG) n , (CGG⅐CCG) n , or (GAA⅐TTC) n , referred to as (CTG) n , (CGG) n , and (GAA) n , respectively (reviewed in Refs. 1-4). These diseases fall into two categories (1).The first, which includes spinal and bulbar muscular atrophy, Huntington's disease, spinocerebellar ataxia type 1, dentatorubral-pallidoluysian atrophy, and Machado-Joseph disease, is characterized by small expansions of a (CTG) n repeat from the ϳ10 -40 in the normal population to ϳ40 -120 units in diseased individuals that encode a polyglutamine tract in the corresponding gene products. This mutation may impart a gain of function to the mature polypeptides that is deleterious to neuronal activity (1-3).The second, which includes the myotonic dystrophy (dystrophia myotonica (DM)) and the fragile X and E (FRAXA and FRAXE) genes, is characterized by much larger expansions of either a (CTG) n or (CGG) n repeat, respectively, in the untranslated region of the genes. The mechanisms by which these expansions lead to disease are not fully understood (5, 6). The number of repeats is polymorphic in the normal population and ranges from 6 to 5...

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Section: Resultsmentioning

confidence: 99%

Triplet Repeat Instability and DNA Topology: An Expansion Model Based on Statistical Mechanics

Gellibolian

Bacolla

Wells

1997

Journal of Biological Chemistry

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“…An experimental determination of an effective bending stiffness of linear DNA has been made from sedimentation data (20). It may be possible to determine the torsional stiffness C from existing data on the free energy of supercoiling (16,21). The present theory also suggests methods for evaluating C either from Eq.…”

Section: Discussionmentioning

confidence: 99%

Elastic model of supercoiling.

Benham¹

1977

Proc. Natl. Acad. Sci. U.S.A.

117

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An elastic model for the supercoiling of duplex DNA is developed. The simplest assumptions regarding the elastic properties of double-helical DNA (homogeneous, isotropic, of circular cross section, and remaining straight when unstressed) will generate two orders of superhelicity when stressed. Recent experimental results [Brady, G. W., Fein, D. B. & Brumberger, H. (1976) Nature 264, 231-2341.suggest that in supercoiled DNA molecules there are regions where two distinct orders of supercoiling arise, as predicted by this model. The role of elasticity in supercoiling of DNA is investigated by ascribing to the double helix the properties of a homogeneous, isotropic elastic rod. The unstressed molecule has helical twist of one turn per ten base pairs. As an elastic object, this idealized molecule resists deviations from straightness and from its unstressed twist with certain bending and torsional stiffnesses.Linear DNA with free ends may deform so as to relieve internal stresses. In its covalently closed, circular form, however, the molecule experiences torsional and bending stresses which cannot be relieved without breaking one of the strands (nicking) (2). Stresses may be varied by external constraints such as dye intercalation (3).If the stresses involved are not too great, the molecule may respond as a whole by deforming into an equilibrium shape. (In this paper "equilibrium" refers to elastic, not thermodynamic, equilibrium.) However, if the stresses are sufficiently large, they may cause local denaturation (4, 5). Disruption of the double helix alters or destroys the elastic properties of the denatured regions, thereby permitting the relief of excess torsional stress through winding or unwinding. In this case, the regions of double helix between adjacent denatured portions deform into equilibrium shapes in response to the stresses imposed on them.The equilibrium shapes possible in these situations may be found from Kirchhoff's theory of linear elastic rods (6). This allows explicit calculation of shapes as desired. It also permits a suggestive visualization of the possible shapes through a rigorous analogy between deforming rods and spinning tops.In summary, it is found that three equilibrium shapes are possible. If the stresses are sufficiently small, a section of double helix may remain straight (neglecting kinetic effects). If the straight shape is not possible, then the double helical sections of the idealized molecule (which may be the whole molecule) will deform either into a (super) helix (a first order supercoil) or into a shape having two orders of superhelicity. Deformation into a (super) helix requires very precise conditions on the imposed loads, and is thereby unlikely to occur in practice.Recent experimental evidence (1) suggests that in supercoiled DNA are found regions exhibiting two orders of superhelicity, as predicted by the theory. This suggests that the elastic model may be adequate to predict supercoiling in DNA. ELASTIC THEORY Two cases are considered here-elastic deformations of ...

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“…Such a test requires an assay that depends on rotation of the DNA during the lifetime of the nicked intermediate. Thus, temperature shifts were used to alter the winding angle of the DNA helix (20,21), and we asked whether the bound topo70 2XCys was capable of relaxing the resultant supercoils in the pRc͞CMV DNA.…”

Section: Analysis Of Sucrose Gradient Fractions For the Presence Of Dna-mentioning

confidence: 99%

DNA relaxation by human topoisomerase I occurs in the closed clamp conformation of the protein

Carey

Schultz

Sisson

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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In cocrystal structures of human topoisomerase I and DNA, the enzyme is tightly clamped around the DNA helix. After cleavage and covalent attachment of the enzyme to the 3 end at the nick, DNA relaxation requires rotation of the DNA helix downstream of the cleavage site. Models based on the cocrystal structure reveal that there is insufficient space in the protein for such DNA rotation without some deformation of the cap and linker regions of the enzyme. Alternatively, it is conceivable that the protein clamp opens to facilitate the rotation process. To distinguish between these two possibilities, we engineered two cysteines into the opposing loops of the ''lips'' region of the enzyme, which allowed us to lock the protein via a disulfide crosslink in the closed conformation around the DNA. Importantly, the rate of DNA relaxation when the enzyme was locked on the DNA was comparable to that observed in the absence of the disulfide crosslink. These results indicate that DNA relaxation likely proceeds without extensive opening of the enzyme clamp. D NA topoisomerases are ubiquitous and essential enzymes that solve the topological problems accompanying key nuclear processes such as DNA replication, transcription, repair, and chromatin assembly by introducing temporary single-or double-strand breaks in the DNA (1-4). In addition, these enzymes fine-tune the steady-state level of DNA supercoiling to facilitate protein interactions with DNA and to prevent excessive supercoiling that is deleterious. There are two fundamental types of topoisomerases, which differ in both mechanism and cellular function (1). Type II enzymes are dimeric, promote the passage of one region of duplex DNA through a double-stranded break in the same or a different molecule, and are primarily dedicated to such processes as DNA supercoiling and chromosome segregation. Type I enzymes are monomeric and transiently break one strand of the duplex DNA, allowing for adjustments in helical winding. These enzymes are primarily responsible for removing torsional stress generated by processes that leave the DNA overwound or underwound. The type I topoisomerases have been further divided into two subfamilies based on sequence comparisons and reaction mechanisms (2). The type IA subfamily is characterized by covalent attachment of the enzyme to the 5Ј end of the broken strand in the nicked intermediate, whereas all members of the type IB subfamily attach to the 3Ј end of the broken strand. The prototype of the type IB subfamily, human topoisomerase I, catalyzes changes in the superhelical state of duplex DNA by transiently breaking one strand of the DNA to allow rotation of one region of the duplex relative to another region (5). Strand cleavage is achieved by the nucleophilic attack of the active site tyrosine on a DNA phosphodiester bond. The resulting formation of a phosphodiester bond between the tyrosine and the 3Ј end of the cleaved strand enables the enzyme to reseal the DNA by simple reversal of the cleavage reaction (1).Human topoisomerase I is a 765...

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Action of nicking-closing enzyme on supercoiled and nonsupercoiled closed circular DNA: formation of a Boltzmann distribution of topological isomers.

Cited by 361 publications

References 16 publications

Triplet Repeat Instability and DNA Topology: An Expansion Model Based on Statistical Mechanics

Triplet Repeat Instability and DNA Topology: An Expansion Model Based on Statistical Mechanics

Elastic model of supercoiling.

DNA relaxation by human topoisomerase I occurs in the closed clamp conformation of the protein

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