Structure and Function of the Bacterial Genome. Charles J. Dorman

Figure 1.15 The topological consequences of transcription. This is the twin supercoil domain model, as proposed by Liu and Wang (1987) and supported by numerous independent experiments. Core RNA polymerase is engaged in transcript elongation and the mRNA is simultaneously being translated by polyribosomes to produce nascent peptides. As the coupled transcription‐translation complex moves from left to right, the DNA template ahead becomes overwound, or positively supercoiled, while the DNA behind becomes underwound, or negatively supercoiled. This situation will bring transcription to a halt as the machinery jams. This is because: (a) the domains of supercoiled DNA cannot be resolved by supercoil lateral diffusion because the transcribed region is bounded by topological barriers (solid black discs) and (b) the bulky transcription‐translation complex cannot rotate around the DNA to relieve the torsional tension in the DNA duplex. The solution to the problem is provided by the DNA‐relaxing activities of topoisomerases: DNA gyrase removes the positive supercoils by introducing negative supercoils, while the negatively supercoiled domain is relaxed by DNA topoisomerase I and IV. Interference with these relaxation processes can result in undesirable outcomes, such as R‐loop formation (Figure 1.16). Topological barriers can be created by head‐to‐head transcription collisions and by collisions between converging replisomes and RNA polymerases; they can also arise from the presence of nucleoprotein complexes and distortions (e.g. sharp bends) in the DNA duplex.

A covalently closed, circular duplex DNA molecule that is neither overwound nor underwound is said to be topologically relaxed. If this circular duplex undergoes an increase in its linking number (overwinding) or a decrease (underwinding) it retains an identical nucleotide sequence compared with the relaxed form but differs from this form topologically (Sinden 1994). For this reason, the relaxed, overwound, and underwound isomeric forms of the circular duplex are referred to as topoisomers of the same DNA molecule. Enzymes that produce topological changes in DNA by altering the linking number are called topoisomerases and E. coli has four: topoisomerase I (topo I), DNA gyrase (a topo II family member), topoisomerase III (topo III), and topoisomerase IV (topo IV) (Bates and Maxwell 2005; Wang 2002) (Table 1.1).

Table 1.1 The topoisomerases of E. coli.

      ^a Type I enzymes change the linking number of the duplex DNA substrate in steps of 1 (ΔLk = 1) while type II enzymes change the linking number in steps of 2 (ΔLk = 2).

1.28 DNA Topoisomerases: DNA Gyrase

Topoisomerases are classed as type I if they change the linking number of DNA in steps of one, and as type II if the linking number changes in steps of two (Champoux 2001; Wang 2002) (Table 1.1). DNA gyrase is a type II enzyme and it has the property, unique to prokaryotes, of being able to introduce negative supercoils into DNA (Gellert et al. 1976a; Higgins et al. 1978; Nöllmann et al. 2007). This negative supercoiling activity is ATP dependent and there is an ATP‐binding site in the B subunit of gyrase (Gellert et al. 1979; Mizuuchi et al. 1978). Gyrase is an A₂B₂ hetero‐tetramer and it is essential; knockout mutations in either of the genes that encode its A (gyrA) or B (gyrB) subunits are lethal (Bates and Maxwell 2005). Its essentiality has made gyrase a very attractive target for antimicrobial therapy and a number of drugs are available that target its subunits (Collin et al. 2011; Maxwell 1999). The coumarin class of antimicrobials have been particularly useful as research tools because they compete with ATP for access to the B subunit ATPase and inhibit gyrase activity without inducing the SOS response (DeMarini and Lawrence 1992; Gellert et al. 1976b; Gormley et al. 1996; Pugsley 1981; Sugino and Cozzarelli 1980; Sugino et al. 1978). In contrast, those drugs (e.g. quinolones) that inhibit the A subunit during DNA cleavage and religation cause DNA damage that results in induction of the SOS response (Gellert et al. 1977), something that can complicate experimental design and data interpretation. Gyrase also has an ATP‐independent DNA relaxing activity that is unmasked only in the absence of ATP (Gellert et al. 1977; Higgins et al. 1978; Williams and Maxwell 1999). The ATP‐dependent mechanism by which gyrase introduces negative supercoils into DNA is also capable of relaxing positive supercoils (Ashley et al. 2017). This activity is especially important when gyrase processes the positively supercoiled DNA that is a by‐product of transcription and DNA replication (Koster et al. 2010) (Figures 1.14 and 1.15). DNA gyrase in living cells responds to the [ATP]/[ADP] ratio, linking the management of DNA topology to the metabolic activity of the bacterium (Hsieh et al. 1991a,b; Snoep et al. 2002; van Workum et al. 1996). Gyrase activity is also tuned in living bacteria by stresses such as the acidification of the bacterial cytosol that accompanies adaptation to acid stress (Colgan et al. 2018).

1.29 DNA Topoisomerases: DNA Topoisomerase IV

Topo IV was discovered in E. coli 14 years after gyrase, its type II topoisomerase companion (Kato et al. 1990) (Table 1.1). It is encoded by two genes, parC and parE, whose names hint at a defect in chromosome partitioning that is associated with mutants with a topo IV deficiency (Kato et al. 1990). Topo IV is an ATP‐dependent topoisomerase but, unlike gyrase, which it closely resembles in amino acid sequence and subunit structure, it cannot introduce negative supercoils into DNA. Instead, Topo IV relaxes both positively and negatively supercoiled DNA and is an important DNA decatenase (Bates and Maxwell 2007; Crisona and Cozzarelli 2006; Kato et al. 1992; Peng and Marians 1993; Zawadzki et al. 2015). Its relationship with the MukBEF SMC‐like complex is emerging as one of Topo IV's most physiologically significant functions, one that is important for the

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