| This dissertation focuses on two basic objectives: first, defining the mechanism by which IN recognizes and catalyzes reactions with viral DNA and second, engineering novel enzymatic activities into IN. Chapter 2 presents an extensive study of the nucleic acid determinants of substrate recognition, which fulfills the first objective. Our hypothesis was: Incorporating modified nucleotides into model viral substrates can elucidate the determinants of specificity. We are the first to present that the conserved position 4-G contains essential minor and major groove interactions with IN important for substrate recognition and catalysis. Amino acid determinants located within IN modulate DNA recognition. The hypothesis was: The origins of IN specificity can be determined by recombining the HHCC, catalytic core, and C-terminal domains in trans (Chapter 4). In a preliminary study, each domain was expressed and purified. The ability of these domains to act in trans remains untested, and will comprise the future work.; The hypothesis of the second part of my dissertation research, fulfilling the second dissertation objective was: Engineering integrase to recognize a specific DNA site in the human genome can be realized by tethering a sequence-specific DNA binding protein to IN. A model system was designed to test site-specific gene transfer into the Epstein-Barr virus (EBV) genome (chapter 3). As an important step toward this goal, I have shown that IN can be fused with the DNA binding and dimerization domains of BZLF-1 with retention of catalytic activity. In addition, these fusion proteins bind specifically to target DNA and thus may be used to target integration. To improve fusion protein purification, a eukaryotic expression system for the production of IN in Pichia pastoris was developed (Chapter 5). The hypothesis was: As a eukaryote, Pichia will express a more soluble, stable, and active IN at greater quantities than E. coli. We are the first to present expression and purification of IN in Pichia pastoris where the protein was produced at a level three fold higher than in E. coli. By producing active, soluble IN, within Pichia a higher quality protein can be available for inhibitor and additional mechanistic and structural studies. |
