dif Site-Specific Recombination and Chromosome Segregation

Leslie, Nicholas Reid (1994) dif Site-Specific Recombination and Chromosome Segregation. PhD thesis, University of Glasgow.

Abstract

Linear chromosomes, for example those of eukaryotes, have evolved mechanisms to ensure that chromosome ends are protected from exonucleolytic attack and can be replicated completely. Circular chromosomes are not subject to these problems. However, their circularity makes homologous recombination a threat to their integrity and maintenance. Any number of homologous recombination events between linear homologous chromosomes generates linear products that have the same overall structure as their parents. In contrast, any odd number of homologous exchanges between circular chromosomes generates a fusion of the two circles. Such dimeric molecules might create difficulties in segregation at cell division, or in packaging when the circles are viral. Clearly, a unit copy replicon in dimeric form can not be normally partitioned into two daughter cells. However, multimerisation also interferes with the stable inheritance of high copy number plasmids. It is therefore not surprising to find that circular genomes have evolved mechanisms to ensure that multimers can be effectively converted to monomers. In Escherichia coli and related bacteria, we believe that both plasmids and the bacterial chromosome use site-specific recombination to convert multimers to monomers. The replication terminus region of the E. coli chromosome encodes a locus, dif, that is required for normal chromosome segregation at cell division, dif is a substrate for site-specific recombination catalysed by the related chromosomally encoded recombinases XerC and XerD. It has been proposed that this recombination converts chromosome multimers formed by homologous recombination back to monomers in order that they can be segregated prior to cell division. Strains mutant in dif, xerC, or xerD, share a characteristic phenotype, containing a variable fraction of filamentous cells with aberrantly positioned and sized nucleoids. It is shown that the only DNA sequences required for wild-type dif function in the terminus region of the chromosome are contained within 33 bp known to bind XerC and XerD, and that certain active site residues of the Xer proteins known to be involved in the catalysis of recombination are required for normal chromosome segregation. It is also shown that recombination by the loxP/Cre system of bacteriophage PI will suppress the phenotype of a dif deletion strain when loxP is inserted in the terminus region. Since neither the dif/Xer, nor the loxP/Cie system caused this suppression when located in other positions on the chromosome, close to oriC or within lacZ, this reinforces the idea that site-specific recombination must occur in the terminus region in order to allow normal chromosome segregation.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Dave Flint
• Keywords: Genetics
• Date of Award: 1994
• Depositing User: Enlighten Team
• Unique ID: glathesis:1994-74676
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 27 Sep 2019 17:13
• Last Modified: 27 Sep 2019 17:13
• URI: https://theses.gla.ac.uk/id/eprint/74676