The tyrosine recombinases XerC and XerD function in the monomerisation of circular dimer replicons in many bacteria. The recombining complex contains two synapsed recombination sites and two molecules each of XerC and XerD. Recombination proceeds through two sequential steps of DNA strand exchanges separated in time and space. A specific pair of recombinases initiates the reaction forming a Holliday junction intermediate, which undergoes a conformational change to allow resolution to recombinant products by the other pair of enzymes. In an attempt to understand the molecular basis of recombination machine assembly and coordination of catalysis, chimeras of XerC and XerD were constructed and their properties studied in partial and complete recombination reactions. XerC and XerD are two-domain proteins, whose C-terminal regions contain all of the catalytic residues. It is demonstrated here that XerC or XerD variants lacking their N-terminal domains are active in recombination when combined with their wild type partners. However, the normal pattern of catalysis is dramatically altered: strand exchange by the recombinase variant is stimulated, while that by the wild type partner is impaired. The primary determinants for the mutant phenotype are shown to reside in the region of a-helix B of XerCD. It is also demonstrated that the exchange of the extreme C-termini of XerCD has a profound effect on the direction of HJ resolution. These observations confirm the importance of the cyclic C-terminal "donor-acceptor" interactions between XerC and XerD. Finally, the recombination reaction catalysed by ResD, a tyrosine recombinase encoded by the F-plasmid of E. coli, which is believed to function in the monomerisation of F-plasmid dimers, was reconstituted in vitro. Recombination is intramolecular and shows topological selectivity. ResD lacks a region corresponding to the N-terminal domains of XerCD, and hence its characterisation might supply further insights about the roles of the N-terminal domains of tyrosine recombinases.

Graduation date: 1993

This thesis reports the synthesis and copper coordination chemistry of covalently-linked aryl-imidazole derivatives designed as models for the crosslinked imidazole-phenol sidechains of the His-Tyr cofactor in the CcO. Three new imidazole- (HL1 - HL3) and three new indole- (HL4 - H2L6) containing tripodal ligands were synthesised. The conjugate addition of an imidazole to activated quinone derivatives was developed as a new route to organic models for the Tyr His cofactor. Two monodentate imidazole-aryl, Im-hq(OH)2 and Im-ArOH, and an imidazole-quinone, Im bq were obtained using this route. The X-ray crystal structure of Im-hq(OH)2.EtOH was determined. The route was also used to give new chelating ligands, H2L10 and HL12, containing a cross-linked imidazole-phenol surrogate for the Tyr244-His240 cofactor. Copper complexes of Im-hq(OH)2, Im-bq, Im-ArOH, H2L10-HL12, and HL1-H2L6 were prepared, and the X-ray crystal structures of [Cu(terpy)(Im-bq)][BF4]2 and five other copper complexes were determined. The physiochemical properties of the copper complexes were characterized by FT-IR, UV-Vis-NIR, EPR and (spectro)electrochemical studies. Key results include: the oxidation of Im-ArO- anion affords the semiquinone radical, Im-sq(4OH)(1O??????), in a hydrous solvent. However, the oxidations of neutral Im-ArOH and [Cu(tpa)(Im-ArOH)]2+ produce the corresponding phenoxy radical species that rapidly and reversibly dimerise to give quinol cyclohexadienone, QCHD, dimers. Significantly [Cu(tpa)(Im-sq(4OH)(1O??????))]2+ was EPR silent, perhaps due to antiferromagnetic coupling between the Cu(II) (S=1/2) and semiquinonyl radical (S=1/2) centres. Deprotonation of the hydroquinone in [Cu(tpa)(Im-hq(OH)2]2+ produces the hydroquinone dianion which reduces the Cu(II) centre. The semiquinone radical is coordinatively labile and dissociates from the Cu(I) centre. The biological implications of these results are mentioned.

Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2001? / Copy of author's previously published work inserted. Includes bibliographical references (leaves 160-182).

Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 2001. / Bibliography: leaves 139-165.

Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 2005? / "October 2004" Includes bibliographical references (leaves 201-256).

Thesis (Ph. D.)--Northeastern University, 2008. / Title from title page (viewed April 2, 2009). Graduate School of Arts and Sciences, Dept. of Chemistry and Chemical Biology. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2008. / Title from first page of PDF file. Includes bibliographical references (p. 114-127).

Thesis (Ph. D.)--Ohio State University, 2003. / Title from first page of PDF file. Document formatted into pages; contains xviii, 158 p. : ill. (some col.). Advisor: Young C. Lin, Veterinary Biosciences Graduate Program. Includes bibliographical references (p. 148-158).

Thesis (M. Phil.)--University of Hong Kong, 1999. / Includes bibliographical references (leaves 75-82).