Two-component homologous recombination systems of viral/phage origin are widely distributed in the microbial world. They include an alkaline exonuclease and partnering single stranded DNA binding protein (SSAP). The exonuclease digests linearized double stranded DNA (dsDNA) molecules generating partially or fully single stranded DNA (ssDNA) intermediates. The SSAP coats these nascent ssDNA molecules and initiates the recombination process by mediating strand annealing and displacement events with homologous DNA targets. The bacteriophage lambda Exo exonuclease and Bet SSAP proteins, and the RecE exonuclease and RecT SSAP proteins from the Escherichia coli Rac prophage are the best-studied two-component homologous recombination systems. Both systems have been utilized in versatile recombination-mediated genetic engineering (‘recombineering’) procedures for the in vivo modification of bacterial DNA. Homologues of the putative YqaJ protein from Bacillus subtilis share homology with Lambda-Exo, and are predicted to function as alkaline exonucleases. However, no YqaJ homologue has yet been functionally characterized, and this hypothetical protein family remains to be formally defined. In the first part of my thesis, I describe the bioinformatic analysis and functional characterization of the YqaJ protein (KO-YqaJ) from a strain of Kingella oralis, a commensal species of oral Neisseriaceae. Multiple sequence alignments indicate that YqaJ homologues (including KO-YqaJ) are ca. 300-340aa in length, with all the highly-conserved residues putatively essential for exonuclease activities located in the ca. 200aa N-terminal region. Phylogenetic analysis indicates that YqaJ exonucleases constitute a separate clade, within the Lambda-Exonuclease (LE) family of exonuclease proteins of phage/viral origin, with inferred evolutionary origins distinct from that of ‘Exo-like’ exonucleases, which are generally ca. 200-220aa in length. The biochemical activities of recombinant KO-YqaJ were established. Results demonstrated that it digests linear dsDNA in the 5’-to-3’ direction in a highly processive manner, with activities dependent on Mg(II) or Mn(II) ions. It prefers dsDNA substrates with 5’-phosphorylated termini, and its exonuclease activities are optimal at pH 8.2 and at 49°C. It has single strand DNA (ssDNA) exonuclease, weak ssDNA endonuclease activities but negligible dsDNA endonuclease activities under the conditions tested. Size-exclusion chromatography revealed that KO-YqaJ forms a stable dimeric arrangement, in contrast to the toroidal trimers formed by Lambda-Exo. Mutational analysis of putative active site residues in KO-YqaJ indicated that Arginine-8 (putatively involved in binding 5’-phosphorylated DNA ends); Glutamate-55, Aspartate-90 and Glutamate-104 (putatively involved in metal ion binding); and Glutamine-131 (which putatively mediates nucleotide binding interactions) played roles that were essential for dsDNA hydrolysis. Truncation analysis demonstrated that the C-terminal region of ca. 100aa, which is absent in ‘Exo-like’ exonucleases, was essentially required for activity. The biological activities of KO-YqaJ and its cognate SSAP (KO-RecT) were evaluated in E. coli cells. The combined ability of KO-YqaJ and KO-RecT to mediate homologous DNA recombination between a PCR-generated dsDNA ‘targeting cassette’ with a homologous region on the E. coli chromosome was scored. Results clearly showed that the KO-YqaJ/RecT two-component homologous recombination system can mediate homologous recombination events in vivo, indicating that it has the potential for use in recombineering applications within oral Neisseriaceae other bacterial systems. / published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Microbiology / Master / Master of Philosophy

published_or_final_version / abstract / toc / Microbiology / Master / Master of Philosophy