Topoisomerase

DNA topoisomerases (or topoisomerases) are enzymes that catalyze changes in the topological state of DNA, interconverting relaxed and supercoiled forms, linked (catenated) and unlinked species, and knotted and unknotted DNA. Topological issues in DNA arise due to the intertwined nature of its double-helical structure, which, for example, can lead to overwinding of the DNA duplex during DNA replication and transcription. If left unchanged, this torsion would eventually stop the DNA or RNA polymerases involved in these processes from continuing along the DNA helix. A second topological challenge results from the linking or tangling of DNA during replication. Left unresolved, links between replicated DNA will impede cell division. The DNA topoisomerases prevent and correct these types of topological problems. They do this by binding to DNA and cutting the sugar-phosphate backbone of either one (type I topoisomerases) or both (type II topoisomerases) of the DNA strands. This transient break allows the DNA to be untangled or unwound, and, at the end of these processes, the DNA backbone is resealed. Since the overall chemical composition and connectivity of the DNA do not change, the DNA substrate and product are chemical isomers, differing only in their topology.

The first DNA topoisomerase was discovered in bacteria by James C. Wang in 1971 and was initially named ω (omega) protein; it is now called Escherichia coli (E. coli) topoisomerase I (topo I) and is a representative of the type IA family of enzymes. Subsequently, a similar activity was found in eukaryotic cells (rat liver) by James Champoux and Renato Dulbecco; the enzyme responsible, eukaryotic topo I, has a distinct mechanism and is representative of the type IB family. The first type II topoisomerase to be discovered was DNA gyrase from bacteria, by Martin Gellert and coworkers in 1976, and also characterized by Nicholas Cozzarelli and co-workers. DNA gyrase catalyzes the introduction of negative supercoils into DNA and is the only type II enzyme to do this, all the others catalyze DNA relaxation. Type II enzymes are mechanistically distinct from type I in being ATP-dependent and transiently cleaving both DNA strands rather than just one. Type II topoisomerases were subsequently identified from bacterial viruses and eukaryotes. Topo EC-codes are as follows: ATP-independent (type I), EC 5.6.2.1; ATP-dependent (type II): EC 5.6.2.2. The exception among the type I topoisomerases, reverse gyrase, which contains a helicase domain (EC 3.6.4.12) and introduces positive supercoiling in an ATP-dependent manner. Therefore it is the sole type I topoisomerase classified as EC 5.6.2.2 (Table 1).

The double-helical structure of DNA involves the intertwining of the two polynucleotide strands around each other, which potentially gives rise to topological problems. DNA topology refers to the crossing of the two DNA strands that alters the twist of the double helix and gives rise to tertiary conformations of DNA, such as supercoils, knots and catenanes. Potential topological issues associated with the double-helical structure of DNA were recognized soon after its structure was first elucidated in 1953 by James Watson, Francis Crick and Rosalind Franklin and developed further by the work of Max Delbruck and John Cairns. Closed-circular double-stranded DNA can be described by 3 parameters: Linking number (Lk), Twist (Tw) and Writhe (Wr) (Fig. 1). Where Lk refers to the number of times the two strands are linked, Tw refers to the number of helical turns in the DNA, measured relative to the helical axis, and Wr quantifies the coiling of the path of the DNA helix in space and is often equated with 'supercoiling'.

The 3 parameters are related as follows: Lk = Tw +Wr. This is a mathematical identity originally obtained by Călugăreanu in 1959 and is referred to as the Călugăreanu, or Călugăreanu–White–Fuller, theorem. Lk cannot be altered without breaking one or both strands of the helix; Tw and Wr are interconvertible and depend upon the solution conditions. Supercoiling is a vernacular term for DNA with a non-zero linking difference, more correctly referred to as specific linking difference (σ = ΔLk/Lk⁰, where Lk⁰ is the mean linking number of the relaxed DNA circle). DNA is said to be positively supercoiled if Lk of it is higher than Lk⁰ for the relaxed state (Lk-Lk^o = ΔLk, ΔLk>0); that means that Tw and/or Wr are increased relative to the relaxed molecule. Conversely, DNA is negatively supercoiled if Lk of the molecule is lower than the Lk⁰ (ΔLk<0).

The consequences of topological perturbations in DNA are exemplified by DNA replication during which the strands of the duplex are separated; this separation leads to the formation of positive supercoils (DNA overwinding or overtwisting) ahead of the replication fork and intertwining of the daughter strands (precatenanes) behind (Fig. 2). If the positive supercoils are not relaxed, progression of the replication fork is impeded, whereas failure to unlink the daughter strands prevents genome segregation, which is required for cell division. Transcription by RNA polymerase also generates positive supercoiling ahead of, and negative supercoiling behind, the transcriptional complex (Fig. 2). This effect is known as the twin-supercoiled domain model, as described by Leroy Liu and James Wang in 1987. These topological perturbations must be resolved for DNA metabolism to proceed, allowing the cell to efficiently replicate, transcribe and partition the genome to enable cellular division and vitality. Knots in DNA can be found in bacteriophages and as products of recombination reactions. In general, knots in DNA are detrimental and need to be removed (by topoisomerases). DNA catenanes are formed upon the replication of circular molecules and need to be resolved by topoisomerases or recombinases to allow proper separation of daughter molecules during cell division. In addition to the detrimental aspects of DNA topology that require resolution, there are also beneficial aspects. For example, plasmid replication requires negative supercoiling of the origin, which facilitates local melting and exposes single-stranded DNA required to initiate replication. Similarly, initiation of replication from the main bacterial origin oriC also requires negative supercoiling. Furthermore, compaction of the E. coli genome is achieved in part by negative supercoiling.

DNA topoisomerases are enzymes that have evolved to resolve topological problems in DNA (Table 2). They do this via transient breakage of one or both strands of DNA. This has led to the classification of topos into two types: type I, which catalyze reactions involving transient single-stranded breaks, and type II, which catalyze reactions involving transient double-stranded breaks (Fig. 3; Table 2). Sub-types exist within these classifications.

These enzymes catalyze changes in DNA topology via transient single-stranded breaks in DNA. Reactions can occur on both single- and double-stranded DNA substrates and can proceed via a 'swivel' or 'strand-passage' mechanism (Fig. 3). The range of reactions includes: DNA supercoil relaxation, unknotting of single-stranded circles, and decatenation, provided at least one partner has a single-stranded region. In the case of the archaeal enzyme, reverse gyrase, positive supercoiling of DNA is possible.

Type IA are monomeric and bind to single-stranded segments of DNA. They introduce a transient single-stranded break through the formation of a tyrosyl-phosphate bond between a tyrosine in the enzyme and a 5′-phosphate in the DNA. The segment of DNA within which the break occurs is called the 'gate' or G-segment, and its cleavage allows the passage of another segment of DNA, the 'transport' or T-segment, to be passed through in a 'strand-passage' process. This is followed by ligation of the G-segment. For strand passage to occur, topo IA must undergo a conformational change to open the DNA gate and allow T-segment transfer. During a DNA relaxation reaction this process changes the linking number of the DNA by +/-1 (Fig. 4). Examples of type IA topoisomerases include prokaryotic topo I and III, eukaryotic topo IIIα and IIIβ and the archaeal enzyme reverse gyrase. Reverse gyrase, which occurs in thermophilic archaea, comprises a type IA topo coupled to a helicase, and is the only known enzyme that can introduce positive supercoils into DNA. The gene encoding reverse gyrase is also found in some groups of thermophilic bacteria, where it was likely transferred by horizontal gene transfer from Archaea.