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Title: The Xer site-specific recombination system
Author: Roberts, Jennifer Ann
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1994
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Three major themes were studied during the course of this work. The role of the XerC binding site in resolution selectivity: Site-specific recombination catalysed by the Escherichia coli proteins XerC and XerD is required for stable inheritance of natural multi-copy plasmids at cell division, and for resolution of chromosome dimers to allow segregation of circular chromosomes. Recombination occurs in both cases at ~30 bp core recombination sites of sinular sequence, which can be divided into a left arm (where XerC binds), a right arm (where XerD binds), and a central region of 6-8 bp between the two arms. Sites that are involved in plasmid resolution, such as cer from plasmid ColE1, also require 220 bp of accessory DNA, and two proteins ArgR (the arginine repressor) and Pep A (aminopeptidase A) to permit recombination to occur. To resolve plasmid dimers, thereby ensuring their heritable stability, site-specific recombination must occur preferentially between sites that are present in direct repeat within a DNA molecule; the molecular mechanisms underlying this "resolution selectivity" were investigated by studying recombination substrates with different left arm sequences. A series of plasmids was constructed varying in sequence at four positions in the left arm, and their abilities to recombine intermolecularly (i.e. without resolution selectivity) was assayed. One base within the left arm was found to be especially important in determining whether or not a site could support intermolecular recombination, but this effect was modified by the other three variant bases in the left arm. Six (of a total of sixteen) sites were analysed further. It was established that XerD bound equivalently to all of the six sites in the absence of XerC, but that the DNA-XerC/D complexes formed in vitro had different electrophoretic mobilities in gel retardation assays; this was proposed to be as a result of differences in protein-induced DNA bending. The relative affinities of XerC/D for the sites also varied depending on left arm sequence. The relative mobilities of the DNA-XerC/D complexes were correlated with both the relative affinities of the sites for XerC/D, and with their abilities or inabilities to support intermolecular recombination. It was therefore suggested that the protein-DNA conformation, and the affinity of the recombinases for a recombination site, could be affected by the sequence of the left arm, and that this affects whether or not a site is effective in resolution of plasmid dimers. The role of Fis on Xer recombination: The role of the DNA-binding protein Fis on Xer recombination was investigated. It was established that recombination at an XerC/D-substrate, dif, was reduced in a fis mutant strain. Fis plays a variety of cellular roles, both in other recombination systems, and as a transcriptional and replication effector, therefore experiments were performed to establish whether the effect of Fis during Xer recombination is exerted directly at the recombination site, or indirectly. The results established that Fis acts indirectly. A fis derivative of ah xerC::mini Mud lacZ strain was constructed, and expression of lacZ in the two strains was compared using P-galatosidase assays. The assays indicated that expression of xerC may be significantly reduced in a fis mutant, implying that Fis might stimulate expression of xerC. A screen for additional xer mutants: A Tn5 mutagensis of the E. coli chromosome was undertaken, with two major aims. We are currently unable to detect products of Xer recombination in vitro, although it is possible to recreate a single strand-exchange either to produce or to resolve Holliday junctions. This has led to concern that there may be additional Xer proteins awaiting discovery, or that the existing reaction conditions are inappropriate. By performing a mutagenesis it was hoped that it would be possible to screen for novel xer mutants not falling into any of the four known complementation groups {argR, pepA, xerC, xerD). A reporter plasmid was constructed for screening of putative mutants, consisting of two psi sites (from plasmid pSClOl) in direct repeat. This plasmid was utilised because recombination at psi was found to be ArgR-independent, but dependent on PepA, XerC and XerD; however, deletion analysis of psi indicated that another protein might be required to bind to this site to perform a similar role to that of ArgR during recombination at cer. The mutagenesis was therefore also undertaken with the second major objective of identifying the protein binding to this site. A mutant strain, JR29, was identified that failed to recombine at psi and at cer, but recombined at dif. JR29 was not complemented by XerC or by XerD, but was partially complemented by PepA. In vivo and in vitro assays also indicated that this strain was a pep A mutant; however, PI transduction of the Tn5 kanamycin resistance allele into another strain failed to produce a pepA mutant, but created a strain, JR30, in which recombination appeared to be at a lower rate. Therefore JR29 appears to contain two mutations: one in pep A, the other in an unidentified locus.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available