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

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1.2). The SeqA protein binds to these sites while they are still in their hemimethylated form, sequestering the origin and excluding DnaA (Han et al. 2003; Slater et al. 1995; von Freiesleben et al. 1994). The sequestered state persists for about one third of the cell cycle when it is relieved by dissociation of SeqA and methylation of the 5′‐GATC‐3′ sites by Dam (Kang, S. et al. 1999; Lu et al. 1994). SeqA also interferes with expression of the dnaA gene, reducing the levels of the DnaA protein available for binding to oriC (Campbell and Kleckner 1990). In addition, SeqA contributes to processes that ensure proper segregation of the chromosome copies at cell division (Helgesen et al. 2015; Waldminghaus and Skarstad 2009). It is interesting to note that both hemimethylated oriC and SeqA have been shown to associate with the cell envelope (Ogden et al. 1988; Slater et al. 1995), perhaps indicating a role for the complex in the positioning of oriC in the cell.

      The converging replication forks moving along the chromosome will create a topological problem as they approach one another in the Ter region. As chromosome replication comes to an end, the products that it generates will emerge as intertwined DNA duplexes. This physical linkage must be resolved if it is not to impede chromosome segregation. Furthermore, if homologous recombination occurs between the sister chromosomes it may create a chromosome dimer. This process is made more likely by oxidative damage to DNA, as occurs in mutants defective in the Fur iron regulatory protein (Steiner and Kuempel 1998). The dimers arise from RecBCD‐ and RecFOR‐mediated events with roughly equal frequency (Barre et al. 2001). Once formed, this dimer must be resolved at or before cell division or an anucleate cell will be created (see Section 1.8).

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      It is not in the interest of the bacterium to have chromosome dimers arising frequently because this poses a risk that either anucleate cells will arise or that the chromosome may be damaged by the division septum as the cell attempts to segregate a dimeric chromosome. RecA‐dependent homologous recombination events, whether they arise via the RecBCD or RecFOR pathways (Section 1.6), generate Holliday junctions that must be resolved by the Ruv resolvasome‐based system. There is a bias in this process in favour of Ruv‐mediated resolution that does not involve crossover, and therefore the creation of chromosome dimers (Cromie and Leach 2000; van Gool et al. 1999). This bias reduces the frequency at which chromosome dimers, and the associated threat to the wellbeing of the daughter cell genomes, arise.

      The XerCD system is versatile and is used in site‐specific recombination reactions with sequences related, but not identical, to dif, and accompanied by co‐factor proteins, to resolve dimers in autonomously replicating plasmids (Clerget 1991; Colloms 2013; Colloms et al. 1990, 1998; Summers 1989). In the human pathogen V. cholerae, the CTXφ bacteriophage that carries the cholera toxin ctxAB operon integrates into chromosome I at its dif site using the XerC recombinase from the V. cholerae XerCD recombinases to catalyse the reaction (it can enter the corresponding location on chromosome II at a lower frequency) (Das et al. 2013; Huber and Waldor 2002; McLeod and Waldor 2004). CTXφ is a filamentous phage and only the plus strand of its genome integrates. The plus strand of the CTXφ genome folds to create a double‐stranded region that encompasses the XerC and XerD binding sites of the phage flanking a mismatched and bulging phage dif site. Only a single stranded exchange occurs, mediated by XerC alone, and this creates a Holliday junction that is resolved by DNA replication (Val et al. 2005). The minus strand of the CTXφ, phage fails to generate a dif site with enough homology to recombine with the chromosomal counterpart, so this strand of the phage genome does not integrate. The integrated phage does not excise because its dif sites also lack sufficient homology with the chromosomal site to promote site‐specific recombination (Val et al. 2005).

      

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