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DNA Supercoiling
DN A S u pe rco i li n g 575 Lk=Lk
DNA Supercoiling D M J Lilley Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.1255
Topological Basis of DNA Supercoiling DNA supercoiling is a special property of circular, double-stranded DNA that is topological in origin. It confers new structural and energetic properties. When the ends of a linear DNA molecule are ligated to produce a covalently closed circle, the two strands become intertwined like the links of a chain, and will remain so unless one of the strands is broken. The number of times one strand is linked with the other is described by a fundamental property of DNA supercoiling, the linking number (Lk). This is related to two geometrical properties of the molecule, the twist (Tw, rotation of the strands about the helical axis) and the writhe (Wr, which measures the path of the helix axis in space). These three properties are related by: Lk Tw Wr
1
A relaxed, closed circular DNA molecule has a linking number (Lk8) given by: Lk N=h
2
where N is the number of base pairs, and h is the helical repeat under the experimental conditions. If a linear DNA molecule with an exact number of turns under the prevailing conditions were ligated into a planar circle in the absence of torsional force, it would have a linking number that equaled the number of turns in the original linear molecule. However, if the circle were closed following the application of a torsional force to the molecule, such that one or more turns was added or subtracted, the resulting over- or underwinding would become trapped in the molecule by the circularization. These molecules would be true isomers of the relaxed species, by virtue of the topology of the molecule, and are called topoisomers. A negatively supercoiled molecule has a linking deficit (Lk) relative to the relaxed species, i.e., Lk Lk
Lk < 0
3
It is often convenient to express the level of supercoiling in the form of a density that is effectively independent of the size of the molecule considered. Superhelix density (s) is given by:
4
Many natural bacterial DNA species are circular and negatively supercoiled. A plasmid extracted from Escherichia coli in mid-exponential growth is typically supercoiled to the extent of s 0.06, although inside the cell the unconstrained supercoiling in general takes about half this value. By contrast, the DNA of some thermophiles is positively supercoiled (overwound, s > 0). In the absence of strand breakage Lk is constant, and therefore the sum of twist and writhe changes is constant in any structural change that maintains strand integrity. Thus, the linking deficit is partitioned between geometric alterations in the molecule of torsional and flexural character: Lk Tw Wr
5
These changes in the shape and geometry of the supercoiled molecule lead to different physical properties, such as sedimentation and frictional properties (Figure 1).
Energetics of DNA Supercoiling Both twisting and writhing deformations are energetically unfavorable, and a supercoiled DNA molecule has a higher free energy compared to its relaxed isomer. The free energy of DNA supercoiling Gsc is quadratically related to the linking number: Gsc 1050 RT=N Lk2
6
where R is the gas constant, T is the absolute temperature, and N is the size of the DNA molecule in base pairs. Thermal fluctuation about a mean linking difference results in a Boltzmann distribution that is Gaussian; this is readily observed by separating the topoisomers of a bacterial plasmid by gel electrophoresis in the presence of an intercalator like chloroquine. The energy held in a supercoiled circle can be substantial. For example, a 4 kb plasmid with a superhelix density of 0.05 has a free energy of supercoiling of 60 kcal mol 1 at 37 8C. Any local perturbation that is underwound relative to B-DNA (such as an unwinding of the helix, or the formation of a cruciform structure or a section of left-handed DNA) contributes a negative twist change that brings about a partial relaxation of the superhelical stress. This reduction in the free energy of supercoiling offsets the free energy of formation of the new DNA structure, and helps stabilize the altered conformation. Since the free energy of supercoiling increases quadratically with linking difference (equation (6)),
576
D N A S up e rco i l i ng sc
rel
Figure 1 Separation of topoisomers of a circular DNA plasmid by gel electrophoresis in polyacrylamide. sc, supercoiled plasmid as isolated from Escherichia coli in exponential growth. Under the electrophoresis conditions, the native distribution of supercoiled topoisomers migrates as a single band. rel, plasmid DNA following partial relaxation with topoisomerase. Each band contains a topoisomer of given linking number, and adjacent bands correspond to Lk 1. The separation is made possible because the physical structure of the topoisomers varies with linking number. there exists a level of negative supercoiling above which the new structure has a stable existence.
Enzymatic Manipulation of DNA Topology Altering the linking number of a topoisomer requires the temporary breakage of at least one strand, and passage of the other through it before resealing. Enzymes called topoisomerases carry out such reactions. These important and ubiquitous enzymes may be classified into two classes, called types I and II. The type I topoisomerases interconvert topoisomers by changes in linking number of 1, while the type II topoisomerases do so by steps of 2; this significant difference underlies a fundamental difference in mechanism of the two classes. All topoisomerases create temporary breaks in the DNA, and the energy of the phosphodiester bond is conserved in the formation of a transient covalent linkage between a DNA
terminus and the protein, usually as a phosphotyrosine linkage. Thus, the topoisomerases are closely related to the site-specific recombinases. Type I topoisomerases relax DNA supercoiling by means of a single-strand break. The linking number is then changed either by allowing a swivel to occur about the nick (eukaryotic enzymes), or by passing the unbroken strand through the break before the phosphodiester linkage is restored (most eubacterial enzymes), thereby leaving a permanent change in the linking number of the DNA circle. No energy transduction is involved in the function of topoisomerase I (excluding reverse gyrase). It is not required for remaking the phosphodiester linkage as the free energy for this is preserved by a temporary covalent DNA± protein linkage, and the position of equilibrium is simply allowed to run `downhill'; i.e., these enzymes just relax supercoiling toward the equilibrium state under the prevailing conditions. The eubacterial topoisomerases I will specifically relax negative supercoiling, while eukaryotic enzymes can relax either negative or positive supercoiling. Type II topoisomerases function by passing duplex DNA through a double-stranded break, thereby altering Lk by steps of 2. DNA gyrase is a type II topoisomerase of E. coli. This enzyme has the special property of coupling the hydrolysis of ATP to the introduction of negative supercoiling into DNA. Thus, DNA gyrase is an A2B2 tetramer, consisting of specialized subunits for topoisomerization and energy transduction. Type II topoisomerases are found in all cells and even some viruses, but DNA gyrase is the only such enzyme that is known to induce negative supercoiling. The balance between the opposing activities of supercoiling (by gyrase) and relaxation (by topoisomerase I) creates a steady-state level of supercoiling, demonstrated in Salmonella by studying mutants in the relevant genes.
DNA Supercoiling and Transcription There is an intimate relationship between dynamic events in DNA and supercoiling. For example, the unwinding of the DNA template required for the initiation of transcription can be strongly affected by the state of supercoiling. Moreover, an elongating RNA polymerase can itself generate DNA supercoiling, particularly where the rotation of the DNA±protein complex is hindered in some way. This is described by the twin supercoiled-domain model of Liu and Wang (1987). Transcription-induced supercoiling is generally well relaxed by cellular topoisomerases, but can be demonstrated very easily in topoisomerase-mutant bacteria.
Do bzh a n s k y, T h e o d o s i us 577 Further Reading
Bates AD and Maxwell A (1993) DNA Topology, pp. 1±114. Oxford: IRL Press. Cozzarelli NR and Wang JC (eds) (1990) DNA Topology and its Biological Effects, pp. 1±480. Plainview, NY: Cold Spring Harbor Laboratory Press. Lilley DMJ, Chen D and Bowater RP (1996) DNA supercoiling and transcription: topological coupling of promoters. Quarterly Review of Biophysics 29: 203±225. Murchie AIH and Lilley DMJ (1992) Supercoiled DNA and cruciform structures. Methods in Enzymology 211: 158±180. Sherratt DJ and Wigley DB (1998) Conserved themes but novel activities in recombinases and topoisomerases. Cell 93: 149±152.
Reference
Liu LF and Wang JC (1987) Supercoiling of the DNA template during transcription. Proceedings of the National Academy of Sciences, USA 84: 7024±7027.
See also: Plasmids; Topoisomerases
DNA Synthesis J Read and S Brenner Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.1861
DNA synthesis is the process whereby deoxynucleic acids (adenine, thymine, cytosine, and guanine) are linked together to form DNA. In vivo, most DNA synthesis occurs as a result of DNA replication but nucleotides can also be incorporated into DNA precursors during repair mechanisms and retroviruses are able to synthesize DNA from viral RNA of virusinfected cells. DNA replication is initiated by the melting of the DNA double helix. Further, local unwinding is catalyzed by the enzyme helicase, which generates regions of single-stranded DNA. The DNA is then primed by the addition of short RNA sequences that provide an initial 30 hydroxyl group to which deoxynucleotides can be added. These primers are later removed. The extension of nucleotide primers requires a group of enzymes called DNA polymerases. Escherichia coli contains DNA polymerases I, II, and III, polymerase III being important for DNA the de novo synthesis of new DNA strands and polymerase I for editing out unpaired strands at the end of the growing strands. The homologous enzymes in animals are polymerases a, b, and g, with a being responsible for nuclear DNA synthesis and g for mitochondrial DNA synthesis. DNA polymerases extend DNA precursors by adding
nucleotides, one at a time, to the 30 end of an RNA/ DNA precursor. This results in the formation of phosphodiester bonds between the 50 phosphate group of one nucleic acid to the 30 hydroxyl group of the next. The type of nucleotide added at each point is determined by Watson±Crick base pairing with the template DNA strand. The efficiency of this process is improved by the 30 ±50 endonuclease activity of DNA polymerases I and II which provides a postsynthetic proofreading mechanism. However, since daughter DNA strands must be synthesized on both strands of the parent DNA, the replication enzymes must move in the 50 ±30 direction on one strand and the 30 ± 50 direction on the other. This problem is solved by synthesizing the leading strand in the 50 ±30 direction in a continuous manner and the lagging strand in the 30 ± 50 direction through the synthesis of short, 50 ±30 Okazaki fragments of DNA. These Okazaki fragments are then connected by the enzyme DNA ligase to form a continuous strand. Thus this mode of DNA synthesis is known as semidiscontinuous replication. DNA can also be synthesized by reverse transcription. This is the mechanism whereby linear duplex DNA is synthesized from a viral RNA precursor in the cytoplasm of virus-infected cells, a process that requires the enzyme reverse transcriptase. Reverse transcription is a useful tool for synthesizing DNA from mRNA precursors in vitro. During this process, an oligonucleotide primer is annealed to the poly(A) tail of the template mRNA. The primer is then extended by the 50 ±30 stepwise addition of nucleotides through the action of reverse transcriptase. The product is a DNA±RNA hybrid, which can be converted into a cDNA by treatment with RNAse and subsequent treatment with DNA polymerase I. See also: DNA Ligases; DNA Polymerases; DNA Structure; Okazaki Fragment; Replication; Reverse Transcription
Dobzhansky, Theodosius R C Lewontin Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.0373
Theodosius Dobzhansky (1900±1975) was responsible for the present understanding of the evolutionary significance of genetic variation within and between populations. His experimental and observational work on the genetics of natural populations, and the generalizations that he made from those observations, established the agendas that still characterize experimental
DNA Supercoiling D M J Lilley Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.1255
Topological Basis of DNA Supercoiling DNA supercoiling is a special property of circular, double-stranded DNA that is topological in origin. It confers new structural and energetic properties. When the ends of a linear DNA molecule are ligated to produce a covalently closed circle, the two strands become intertwined like the links of a chain, and will remain so unless one of the strands is broken. The number of times one strand is linked with the other is described by a fundamental property of DNA supercoiling, the linking number (Lk). This is related to two geometrical properties of the molecule, the twist (Tw, rotation of the strands about the helical axis) and the writhe (Wr, which measures the path of the helix axis in space). These three properties are related by: Lk Tw Wr
1
A relaxed, closed circular DNA molecule has a linking number (Lk8) given by: Lk N=h
2
where N is the number of base pairs, and h is the helical repeat under the experimental conditions. If a linear DNA molecule with an exact number of turns under the prevailing conditions were ligated into a planar circle in the absence of torsional force, it would have a linking number that equaled the number of turns in the original linear molecule. However, if the circle were closed following the application of a torsional force to the molecule, such that one or more turns was added or subtracted, the resulting over- or underwinding would become trapped in the molecule by the circularization. These molecules would be true isomers of the relaxed species, by virtue of the topology of the molecule, and are called topoisomers. A negatively supercoiled molecule has a linking deficit (Lk) relative to the relaxed species, i.e., Lk Lk
Lk < 0
3
It is often convenient to express the level of supercoiling in the form of a density that is effectively independent of the size of the molecule considered. Superhelix density (s) is given by:
4
Many natural bacterial DNA species are circular and negatively supercoiled. A plasmid extracted from Escherichia coli in mid-exponential growth is typically supercoiled to the extent of s 0.06, although inside the cell the unconstrained supercoiling in general takes about half this value. By contrast, the DNA of some thermophiles is positively supercoiled (overwound, s > 0). In the absence of strand breakage Lk is constant, and therefore the sum of twist and writhe changes is constant in any structural change that maintains strand integrity. Thus, the linking deficit is partitioned between geometric alterations in the molecule of torsional and flexural character: Lk Tw Wr
5
These changes in the shape and geometry of the supercoiled molecule lead to different physical properties, such as sedimentation and frictional properties (Figure 1).
Energetics of DNA Supercoiling Both twisting and writhing deformations are energetically unfavorable, and a supercoiled DNA molecule has a higher free energy compared to its relaxed isomer. The free energy of DNA supercoiling Gsc is quadratically related to the linking number: Gsc 1050 RT=N Lk2
6
where R is the gas constant, T is the absolute temperature, and N is the size of the DNA molecule in base pairs. Thermal fluctuation about a mean linking difference results in a Boltzmann distribution that is Gaussian; this is readily observed by separating the topoisomers of a bacterial plasmid by gel electrophoresis in the presence of an intercalator like chloroquine. The energy held in a supercoiled circle can be substantial. For example, a 4 kb plasmid with a superhelix density of 0.05 has a free energy of supercoiling of 60 kcal mol 1 at 37 8C. Any local perturbation that is underwound relative to B-DNA (such as an unwinding of the helix, or the formation of a cruciform structure or a section of left-handed DNA) contributes a negative twist change that brings about a partial relaxation of the superhelical stress. This reduction in the free energy of supercoiling offsets the free energy of formation of the new DNA structure, and helps stabilize the altered conformation. Since the free energy of supercoiling increases quadratically with linking difference (equation (6)),
576
D N A S up e rco i l i ng sc
rel
Figure 1 Separation of topoisomers of a circular DNA plasmid by gel electrophoresis in polyacrylamide. sc, supercoiled plasmid as isolated from Escherichia coli in exponential growth. Under the electrophoresis conditions, the native distribution of supercoiled topoisomers migrates as a single band. rel, plasmid DNA following partial relaxation with topoisomerase. Each band contains a topoisomer of given linking number, and adjacent bands correspond to Lk 1. The separation is made possible because the physical structure of the topoisomers varies with linking number. there exists a level of negative supercoiling above which the new structure has a stable existence.
Enzymatic Manipulation of DNA Topology Altering the linking number of a topoisomer requires the temporary breakage of at least one strand, and passage of the other through it before resealing. Enzymes called topoisomerases carry out such reactions. These important and ubiquitous enzymes may be classified into two classes, called types I and II. The type I topoisomerases interconvert topoisomers by changes in linking number of 1, while the type II topoisomerases do so by steps of 2; this significant difference underlies a fundamental difference in mechanism of the two classes. All topoisomerases create temporary breaks in the DNA, and the energy of the phosphodiester bond is conserved in the formation of a transient covalent linkage between a DNA
terminus and the protein, usually as a phosphotyrosine linkage. Thus, the topoisomerases are closely related to the site-specific recombinases. Type I topoisomerases relax DNA supercoiling by means of a single-strand break. The linking number is then changed either by allowing a swivel to occur about the nick (eukaryotic enzymes), or by passing the unbroken strand through the break before the phosphodiester linkage is restored (most eubacterial enzymes), thereby leaving a permanent change in the linking number of the DNA circle. No energy transduction is involved in the function of topoisomerase I (excluding reverse gyrase). It is not required for remaking the phosphodiester linkage as the free energy for this is preserved by a temporary covalent DNA± protein linkage, and the position of equilibrium is simply allowed to run `downhill'; i.e., these enzymes just relax supercoiling toward the equilibrium state under the prevailing conditions. The eubacterial topoisomerases I will specifically relax negative supercoiling, while eukaryotic enzymes can relax either negative or positive supercoiling. Type II topoisomerases function by passing duplex DNA through a double-stranded break, thereby altering Lk by steps of 2. DNA gyrase is a type II topoisomerase of E. coli. This enzyme has the special property of coupling the hydrolysis of ATP to the introduction of negative supercoiling into DNA. Thus, DNA gyrase is an A2B2 tetramer, consisting of specialized subunits for topoisomerization and energy transduction. Type II topoisomerases are found in all cells and even some viruses, but DNA gyrase is the only such enzyme that is known to induce negative supercoiling. The balance between the opposing activities of supercoiling (by gyrase) and relaxation (by topoisomerase I) creates a steady-state level of supercoiling, demonstrated in Salmonella by studying mutants in the relevant genes.
DNA Supercoiling and Transcription There is an intimate relationship between dynamic events in DNA and supercoiling. For example, the unwinding of the DNA template required for the initiation of transcription can be strongly affected by the state of supercoiling. Moreover, an elongating RNA polymerase can itself generate DNA supercoiling, particularly where the rotation of the DNA±protein complex is hindered in some way. This is described by the twin supercoiled-domain model of Liu and Wang (1987). Transcription-induced supercoiling is generally well relaxed by cellular topoisomerases, but can be demonstrated very easily in topoisomerase-mutant bacteria.
Do bzh a n s k y, T h e o d o s i us 577 Further Reading
Bates AD and Maxwell A (1993) DNA Topology, pp. 1±114. Oxford: IRL Press. Cozzarelli NR and Wang JC (eds) (1990) DNA Topology and its Biological Effects, pp. 1±480. Plainview, NY: Cold Spring Harbor Laboratory Press. Lilley DMJ, Chen D and Bowater RP (1996) DNA supercoiling and transcription: topological coupling of promoters. Quarterly Review of Biophysics 29: 203±225. Murchie AIH and Lilley DMJ (1992) Supercoiled DNA and cruciform structures. Methods in Enzymology 211: 158±180. Sherratt DJ and Wigley DB (1998) Conserved themes but novel activities in recombinases and topoisomerases. Cell 93: 149±152.
Reference
Liu LF and Wang JC (1987) Supercoiling of the DNA template during transcription. Proceedings of the National Academy of Sciences, USA 84: 7024±7027.
See also: Plasmids; Topoisomerases
DNA Synthesis J Read and S Brenner Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.1861
DNA synthesis is the process whereby deoxynucleic acids (adenine, thymine, cytosine, and guanine) are linked together to form DNA. In vivo, most DNA synthesis occurs as a result of DNA replication but nucleotides can also be incorporated into DNA precursors during repair mechanisms and retroviruses are able to synthesize DNA from viral RNA of virusinfected cells. DNA replication is initiated by the melting of the DNA double helix. Further, local unwinding is catalyzed by the enzyme helicase, which generates regions of single-stranded DNA. The DNA is then primed by the addition of short RNA sequences that provide an initial 30 hydroxyl group to which deoxynucleotides can be added. These primers are later removed. The extension of nucleotide primers requires a group of enzymes called DNA polymerases. Escherichia coli contains DNA polymerases I, II, and III, polymerase III being important for DNA the de novo synthesis of new DNA strands and polymerase I for editing out unpaired strands at the end of the growing strands. The homologous enzymes in animals are polymerases a, b, and g, with a being responsible for nuclear DNA synthesis and g for mitochondrial DNA synthesis. DNA polymerases extend DNA precursors by adding
nucleotides, one at a time, to the 30 end of an RNA/ DNA precursor. This results in the formation of phosphodiester bonds between the 50 phosphate group of one nucleic acid to the 30 hydroxyl group of the next. The type of nucleotide added at each point is determined by Watson±Crick base pairing with the template DNA strand. The efficiency of this process is improved by the 30 ±50 endonuclease activity of DNA polymerases I and II which provides a postsynthetic proofreading mechanism. However, since daughter DNA strands must be synthesized on both strands of the parent DNA, the replication enzymes must move in the 50 ±30 direction on one strand and the 30 ± 50 direction on the other. This problem is solved by synthesizing the leading strand in the 50 ±30 direction in a continuous manner and the lagging strand in the 30 ± 50 direction through the synthesis of short, 50 ±30 Okazaki fragments of DNA. These Okazaki fragments are then connected by the enzyme DNA ligase to form a continuous strand. Thus this mode of DNA synthesis is known as semidiscontinuous replication. DNA can also be synthesized by reverse transcription. This is the mechanism whereby linear duplex DNA is synthesized from a viral RNA precursor in the cytoplasm of virus-infected cells, a process that requires the enzyme reverse transcriptase. Reverse transcription is a useful tool for synthesizing DNA from mRNA precursors in vitro. During this process, an oligonucleotide primer is annealed to the poly(A) tail of the template mRNA. The primer is then extended by the 50 ±30 stepwise addition of nucleotides through the action of reverse transcriptase. The product is a DNA±RNA hybrid, which can be converted into a cDNA by treatment with RNAse and subsequent treatment with DNA polymerase I. See also: DNA Ligases; DNA Polymerases; DNA Structure; Okazaki Fragment; Replication; Reverse Transcription
Dobzhansky, Theodosius R C Lewontin Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.0373
Theodosius Dobzhansky (1900±1975) was responsible for the present understanding of the evolutionary significance of genetic variation within and between populations. His experimental and observational work on the genetics of natural populations, and the generalizations that he made from those observations, established the agendas that still characterize experimental