and segregation during the cell cycle (Section 1.21).
1.30 DNA Topoisomerases: DNA Topoisomerase I
The principal source of relaxation activity for negatively supercoiled DNA is Topo I, a monomeric, ATP‐independent enzyme that is encoded by the topA gene in the Ter macrodomain (Margolin et al. 1985) (Table 1.1). This enzyme also has a catenase/decatenase activity on double‐stranded circular DNA with single‐stranded regions (Terekhova et al. 2012; Tse and Wang 1980) and prevents over‐replication of the chromosome originating at oriC (Usongo and Drolet 2014). Topo I is a type I topoisomerase that has a ‘swivelase’ activity. It cuts one of the two DNA strands in a negatively supercoiled molecule, forming a covalent link to the cut strand, and allows the torsional strain in the DNA to drive the rotation of the intact strand through the gap (Kirkegaard and Wang 1978). The result is an increase of 1 in the linking number of the DNA (Bates and Maxwell 2005). The topA gene is not essential, although knockout mutants grow slowly (Margolin et al. 1985; Sternglanz et al. 1981). Bacteria that lose Topo I through topA knockout mutations can compensate in different ways, restoring a growth rate that is close to that of wild‐type strains (Raji et al. 1985; Richardson et al. 1984). One option is to acquire non‐lethal mutations in gyrA or gyrB that result in the expression of a gyrase with a reduced negative supercoiling activity (DiNardo et al. 1982; Pruss and Drlica 1985; Pruss et al. 1982, 1986; Richardson et al. 1984, 1988). Another possibility exploits the amplification of the copy number of the parC and parE genes, resulting in increased expression of Topo IV. In these strains the elevated expression of Topo IV with its DNA‐relaxing activity can compensate for the missing Topo I, restoring the growth rate of the mutant to one that is similar to wild type (Dorman et al. 1989; Free and Dorman 1994; McNairn et al. 1995). Bacteria that are not exposed to stressful growth conditions such as elevated temperature, low pH, or raised osmotic stress or lack of oxygen do not require compensatory mutations in order to display normal rates of growth (Ní Bhriain and Dorman 1993). This suggests that a link exists between environmental stress and the management of DNA supercoiling in bacteria (Dorman and Dorman 2016).
1.31 DNA Topoisomerases: DNA Topoisomerase III
Topo III is a second type I topoisomerase that, like Topo I, is an ATP‐independent monomeric enzyme (Table 1.1). It is encoded by the topB gene and is not essential for the survival of the bacterium (Usongo et al. 2013). However, mutants deficient in both Topo I and in Topo III do not survive (Stupina and Wang 2005). Topo III has weak DNA‐relaxing activity and functions principally as a decatenase (Nurse et al. 2003; Perez‐Cheeks et al. 2012). Its apparent weakness as a DNA‐relaxing enzyme compared with Topo I arises from a difference in the mechanisms used by the two topoisomerases: Topo I operates in a processive manner with short pauses between processive runs, whereas Topo III takes long pauses, leading to a relaxing process with an overall rate that seems slower (Terekhova et al. 2012). While Topo I plays an important role in controlling the frequency of chromosome replication initiation at oriC, Topo III contributes to the management of replication fork collision in the Ter macrodomain (Suski and Marians 2008). In fact, all four topoisomerases are important components of the chromosome replication machinery and display both a division of labour and an interesting degree of redundancy that allows the cell to continue to function even if one of the enzymes experiences interference.
1.32 DNA Replication and Transcription Alter Local DNA Topology
The linking number of DNA is changed at a local level by the processes of transcription and DNA replication. In 1987, Liu and Wang proposed, in a landmark theoretical paper, that the process of transcription would induce overwinding of the DNA template ahead of RNA polymerase and underwinding behind (Liu and Wang 1987) (Figure 1.15). Experimental studies provided support for the proposal, leading to the realisation that topoisomerases play important roles in transcription by relieving the torsional stress that the process creates (Ahmed et al. 2017; Chong et al. 2014; Higgins 2014; Rahmouni and Wells 1992; Rani and Nagaraja 2019; Wu et al. 1988). The role of local DNA supercoiling in the modulation of transcription and in gene‐to‐gene communication will be addressed in Section 8.2. Here we will consider the impact of transcription on nucleoid architecture and on DNA replication.
1.33 Transcription and Nucleoid Structure
Several investigations have made links between patterns of transcription and the superstructure of the bacterial nucleoid. At a practical level, replication fork movement must be reconciled with the needs of transcription (initiation, elongation, and termination), so aligning replisome movement with the direction of gene transcription avoids significant conflicts between DNA and RNA polymerases. Collisions between the replisome and RNA polymerase are known to cause severe inhibition of replisome progression (Mirkin and Mirkin 2005). Transcriptional promoters that oppose the direction of replisome movement serve to pause the replication fork, while transcription terminators that are aligned with the direction of replisome movement also act as replication fork pause sites (Mirkin et al. 2006). Conflicts between the replisome and RNA polymerase can generate R‐loops, stalling replication, and transcription in the affected region until RNase H removes the R‐loop (Kuziminov 2018). Unresolved R‐loops also result in hyper‐recombination and genome instability, so avoiding replication–transcription conflicts is very desirable (Figure 1.16).
Figure 1.16 R‐loop formation. When RNA polymerase reads a G+C‐rich template, stalls and backtracks, it leaves a domain of hypernegatively supercoiled in its wake and the associated transcription stalling may allow the RNA transcript to base pair with its DNA template strand, leaving the non‐transcribed strand as a single‐stranded bubble. Other impediments to RNA polymerase progression include head‐on collisions with other transcription units or with replisomes (the barrier is represented by the vertical gapped line). Loss of topoisomerase I activity is known to promote R‐loop formation because it encourages the accumulation of hyper negative superhelicity in DNA that is being transcribed (or replicated). Failure to process and remove RNA loops can lead to DNA damage, including double‐stranded breaks and hyper‐recombination. The Rho transcription‐terminating helicase (Figure 3.4) helps to suppress R‐loop formation by preventing backtracking by RNA polymerase while RNase H eliminates R‐loops by removing the RNA component of the RNA:DNA hybrid in R‐loops.
A correlation has also been reported between gene essentiality and alignment with the direction of replication fork movement (Rocha and Danchin 2003). Overall, one finds more genes on the leading strand than on the lagging strand of the chromosome and this may reflect the outcome of evolutionary pressure to minimise collisions (Rocha 2008). The question of replication and transcription alignment/collision also has a DNA topological dimension, in that converging polymerases will create, and trap between them, a domain of positively supercoiled DNA that must be resolved by type II topoisomerases: gyrase or topo IV (Crisona et al. 2000; Kato et al. 1992). The mechanisms of action of these enzymes bring an increased risk of double‐stranded breaks occurring in the chromosome with potentially lethal consequences for the cell (Hiasa et al. 1996; Lockshon and Morris 1983). These factors may impose limits on the options for gene orientation on the chromosome, influencing its evolution.
Experiments using chromosome conformation capture (3C) methods (Dekker et al. 2002) have suggested a link between
