Structure and Function of the Bacterial Genome. Charles J. Dorman
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of C. crescentus using ‘carbon copy chromosome conformation capture’ (5C) has shown that the replichores lying in between the cell‐pole‐located Ori and Ter regions are intertwined (Umbarger et al. 2011).
Methods based on different forms of chromosome conformation capture offer the possibility of generating high‐resolution data of the co‐location of different parts of the chromosome at different points in time. This offers the possibility of assembling a four‐dimensional map of the bacterial genome. In contrast, investigations that rely on arrays of binding sites for fluorescently labelled DNA‐binding proteins are limited to information about just those few positions where the arrays have been placed. There may also be technical difficulties that arise if the binding site copies in the array recombine; shortening array length and so reducing the strength of the fluorescent signal as fewer labelled proteins bind. The nucleoprotein complexes at the arrays may impede replication fork passage or interfere with local transcription or the diffusion of DNA supercoils (Le and Laub 2014). It is also possible that the bound proteins may interact with one another, bridging together sister chromosomes prior to chromosome segregation (Mirkin et al. 2014).
The movement of the Ori and Ter domains within the E. coli cytosol is thought to reflect the separation of sister chromosomes that are connected along their length (Bates and Kleckner 2005). The molecular nature of the inter‐sister connections is unknown (Kleckner et al. 2014). The final phase of the replication process copies the Ter region, setting the stage for chromosome segregation (Figure 1.9). In fast‐growing bacteria, another round of chromosome replication will have started by the time this stage is reached (Youngren et al. 2014). The E. coli pattern of chromosome domain positioning with Ori and Ter at mid‐cell is not universal: C. crescentus has its Ori and Ter regions in opposite poles of the cell while B. subtilis oscillates between the two modes (Figure 1.11). The differences are thought to reflect different chromosome segregation mechanisms (Wang et al. 2014).
Decatenation of the interlinked chromosome copies by topoisomerase IV and the resolution of any chromosome multimers by XerCD are needed prior to sister chromosome segregation (Hiraga 1993). In the absence of Topo IV, XerCD‐dif‐FtsK can achieve the same outcome by a process of local reconnection involving multiple rounds of site‐specific recombination (Grainge et al. 2007). Finally, the segregation process will move one chromosome, together with any associated live replication forks, into one of the daughter cells. In E. coli and its close relatives, this occurs without the aid of a dedicated protein‐based active partitioning system equivalent to the ParAB proteins and the parS cis‐acting partitioning DNA sequence that are found in most other bacteria (Badrinarayanan et al. 2015; Bignell and Thomas 2001). Radial confinement of the two sister chromosomes has been proposed as playing a role in segregation in the case of E. coli. Here the two chromosome polymers repel one another through an entropic exclusion mechanism that drives the copies into separate compartments before the closure of the cell division septum (Jun and Wright 2010; Junier et al. 2014).
The specificity of chromosome orientation within the cytoplasm has led to the interesting proposal that the chromosome provides the prokaryotic cell with an internal frame of reference, something that has been lost in eukaryotes because the genetic material there is in a membrane‐enclosed nucleus (Theriot 2013). This reference frame is useful in providing each molecule in the cell with a set of spatial coordinates. Developing the point further, Theriot has proposed that eukaryotes rely on their cytoskeleton to provide a reference frame (Theriot 2013). We will return to the issue of spatial and temporal positioning of molecules (Chapter 8).
1.20 SeqA and Nucleoid Organisation
DNA‐binding proteins play important roles in organising the structure of the folded chromosome, with some of these proteins having a chromosome‐domain‐specific binding pattern (Dame et al. 2011). The SeqA protein was introduced during the description of factors involved in the control of the initiation of chromosome replication (Section 1.3). SeqA accompanies the moving replication fork (Brendler et al. 2000; Onogi et al. 1999), resulting in a dynamic pattern of binding around the chromosome (Sánchez‐Romero et al. 2010; Waldminghaus and Skarstad 2010). This protein seems to be excluded from binding within the Ter macrodomain, possibly reflecting the absence from Ter of matches to the consensus sequence for SeqA high‐affinity binding sites (Sánchez‐Romero et al. 2010; Waldminghaus and Skarstad 2010). The SeqA protein can interact with the cell envelope as well as hemimethylated DNA (D'Alençon et al. 1999; Mika et al. 2015; Shakibai et al. 1998; Slater et al. 1995) so may it play a role in the positioning of Ori during cell division (Dame et al. 2011).
1.21 MukB, a Condensin‐Like Protein
The bacterial chromosome is maintained in an orderly superstructure to facilitate replication, transcription, and other DNA‐based transactions. The family of SMC proteins play an important role in achieving this organisation (Uhlmann 2016). These large polypeptides have a DNA‐binding head domain and long coiled‐coil domains that bring the head‐domain‐DNA complexes together in a condensed nucleoprotein complex (Figure 1.10). The head domains have ATPase activity and a DNA‐binding hinge region in the coiled‐coil domain promotes dimer formation (Chen, N., et al. 2008; Kumar et al. 2017a). SMC activity is found in eukaryotes and in prokaryotes, with one of the best‐studied examples of an SMC‐like protein in bacteria being the MukB protein from E. coli (Niki et al. 1991; Rybenkov et al. 2014).
MukB forms topologically stable loops in the chromosomal DNA and protects the supercoils in these protected loops (Kumar et al. 2017a). It forms a complex with the MukE and MukF proteins, with these seeming to play a role in the turnover of the MukB complex on DNA in combination with the ATPase activity of MukB itself (Kumar et al. 2017a). MukF performs a bridging role between the ATPase heads of the two MukB in the complex (Figure 1.10). Proteins performing this task in SMC complexes are called kleisins. The equivalent system in B. subtilis and C. crescentus consists of the proteins Smc (MukB), ScpA (MukF, kleisin), and ScpB (MukE): the ‘Scp’ designation indicates that the protein is a ‘segregation and condensation protein’ (Britton et al. 1998; Burmann et al. 2013; Jensen and Shapiro 1999, 2003; Mascarenhas et al. 2005).
The MukBEF complex has important architectural and segregational roles in the nucleoid, operating mainly outside the Ter macrodomain of the chromosome. Its principal site of action seems to be at ori and MukB requires MukE, MukF, and ATP hydrolysis to gain and maintain this association; MukBEF/SMC complexes do not seem to track moving replisomes (Badrinarayanan et al. 2012a,b; Danilova et al. 2007; Gruber and Errington 2009; Sullivan et al. 2009). MukBEF is responsible for guiding the newly replicated ori regions into the two halves of the cell, driving bipolar segregation; if MukBEF is removed, the ori shifts from mid‐cell to the pole, disturbing normal chromosome orientation and segregation patterns (Danilova et al. 2007). Inside the Ter macrodomain, the MatP protein prevents MukBEF from playing a structural role by displacing it and so making it available for ori binding (Lioy et al. 2018; Nolivos and Sherratt 2014; Nolivos et al. 2016). The MukBEF complex is not required for sister chromosome cohesion because muk mutants have a higher degree of cohesion of sister chromosomes than wild‐type cells (Danilova et al. 2007).
Despite being able to stimulate Topo IV activity by direct interaction (Hayama and Marians 2010; Hayama et al. 2013; Li et al. 2010; Vos et al. 2013), MukBEF forms a complex with Topo IV that seems to stabilise MukBEF on the DNA and drive chromosome condensation independently of the catalytic activities of the topoisomerase (Kumar et al. 2017b). This observation is indicative of an architectural role for the MukBEF‐Topo‐IV complex. Data from chromosome conformation capture experiments show that, together with