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Abstract
Specialized DNA recombination, DNA transposition and site-specific recombination, creates genetic diversity by altering the genetic information through DNA insertions, deletions, and inversions. The goal of this research was to determine the molecular mechanism by which recombinases of the novel Piv/MooV family mediate specialized recombination. While there are greater than 50 members in the Piv/MooV family, two recombinases were chosen for the focus of this dissertation, Piv (pilin invertase) and MooV (mover of IS492 in oceanic variants). Piv catalyzes conservative site-specific inversion of a 2.1 kb chromosomal segment that encodes type 4 pili. MooV catalyzes movement of the insertion element, IS492, in Pseudoalteromonas atlantica. Site-specific insertion and precise excision of IS492 within epsG, the gene for glucosyl transferase, controls phase variable expression of extracellular polysaccharide (EPS). To aid in the determination of the mechanism of Piv-mediated inversion, DNA sequence requirements at the recombination sites for Piv-mediated recombination were defined. In vitro DNA binding assays showed that Piv preferentially binds to single-stranded DNA instead of double-stranded inv sequence; in addition, this binding appears to be structure specific rather than sequence specific. To address the amino acid sequence requirements for MooV-mediated recombination, individual substitutions were generated within a DEDD motif in MooV which is highly conserved in the Piv/MooV family of recombinases and is essential for Piv-mediated inversion. These MooV variants were assayed in vivo for IS492 excision and insertion; each of the conserved acidic residues was shown to be required for both excision and insertion of IS492. A maltose binding protein MooV fusion protein was purified and used in DNA cleavage and strand transfer assays in order to isolate and characterize intermediates in MooV-mediated recombination; however, no in vitro activity of MooV was detected. Therefore, to improve the in vitro activity of MooV, an assay was designed to isolate hyperactive insertion variants of MooV. This approach is likely to allow the elucidation of the mechanism of MooV-mediated recombination of IS492.