Analysis of xer Site-Specific Recombination In Vivo

McCulloch, Richard (1992) Analysis of xer Site-Specific Recombination In Vivo. PhD thesis, University of Glasgow.

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The xer site-specific recombination system is employed in two related biological processes. The first of these roles is in resolving multimers of high copy number, randomly partitioned plasmids in order to ensure their heritable stability. Plasmid multimers arise through homologous recombination. Their resolution to monomers by xer site- specific recombination requires a recombination locus named cer that was originally described in the naturally occurring plasmid ColEl, although similar sites have been isolated in many other plasmids (Summers and Sherratt, 1984; Summers et al, 1985). The second role of the xer system is in bacterial chromosome partition. A locus has been described, named dif, in the terminus region of the Escherichia coli chromosome that is a substrate for xer catalysed site-specific recombination (Blakely et al, 1991; Kuempel et al, 1991). It is believed xer recombination activity is required at dif because dimers of replicating chromosomes are produced by homologous recombination; these dimers cannot be partitioned into daughter cells as the host cell divides, and hence xer recombination converts the chromosomal dimers into monomers and allows cell division. The proteins that act in xer site-specific recombination are encoded by the E.coli chromosome. At the start of this work three genes {xer genes) had been shown to be absolutely required for cer recombination, and one for dif recombination. xerC has been cloned and sequenced and is required for both cer and dif recombination (Colloms et al, 1990; Blakely et al, 1991). The protein encoded by xerC binds to both recombination sites in vitro and has sequence homologies to the X integrase family of site-specific recombinases. xerA and xerB encode the proteins ArgR and aminopeptidase A (PepA) respectively; both are essential for cer recombination and are believed to be "accessory factors" in the reaction (Stirling et al, 1988 and 1989). While this work was being performed a fourth xer gene was identified, called xprB. Genetic evidence is presented that the XprB is essential for both cer and dif recombination and that it shows amino acid sequence homologies to the X integrase family of recombinases. PepA is an amino-terminal exopeptidase whose role in the cer recombination reaction was not understood at the start of this work. Site-directed mutagenesis of the gene encoding PepA is described, and evidence presented that the mutant enzyme (named E354A) encoded by the altered pepA gene lacks any aminopeptidase activity both in vitro and in vivo but is still able to support cer recombination. The relevance of this observation to how PepA is employed by the xer site-specific recombination reaction is discussed. The construction of an controllable in vivo recombination system and its use in analysing the mechanism of the xer recombination reaction is described. The system comprises a derivative of E.coli K12, named RM40, in which the expression oi xerC is controlled by the lac promoter and operator sequences rather than its natural promoter sequences. Analysing the products derived from cer-mediated recombination of reporter plasmids demonstrated the existence of Holliday junction structures produced during the reaction. These putative recombination intermediates were shown to have arisen by the exchange of a specific pair of strands within the cer sites of the substrate DNA molecule used. These results suggest that cer site-specific recombination involves the same form of strand exchange mechanism that has been described in vitro for other members of the X integrase family (Int, FLP and Cre; Stark et al, 1992). The Holliday junctions were isolated in anomalously large quantities when compared to the isolation of such reaction intermediates in other, related systems. The possible relevance of this to the xer recombination mechanism was analysed by comparing cer and recombination in RM40, and by altering the cer substrate molecules and reaction conditions employed.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Marshall Stark
Keywords: Genetics
Date of Award: 1992
Depositing User: Enlighten Team
Unique ID: glathesis:1992-75014
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 27 Sep 2019 14:36
Last Modified: 27 Sep 2019 14:36

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