EBV episomes replicate once per cellular S phase, during latent infection of host cells. Only one viral protein, Epstein-Barr Nuclear Antigen-1 (EBNA-1) is required for replication; the rest of the replication machinery is provided by the cell. EBNA-1 is an excellent model to study the molecular events required for DNA replication and its regulation because viral replication is limited to once per cell cycle. EBNA-1 is a member of a special class of DNA binding proteins called origin binding proteins (OBPs). These specialized proteins bind to distinct DNA sequences in the genome called origins of replication, where DNA replication is initiated. Origin binding proteins may serve to distort the DNA at the origin and may also attract the cellular replication machinery. Structural studies of the DNA binding and dimerization region of EBNA-1 using X-ray crystallography were undertaken in order to better understand how OBPs bind to origin DNA sequences and facilitate the assembly of the cellular replication apparatus. Six truncation mutants of EBNA-1, all containing the DNA binding and dimerization region of EBNA-1, were cloned, over-expressed in bacteria and purified to apparent homogeneity. Four of these clones were crystallized using the method of hanging-drop vapour-diffusion. Two fragments, EBNA₄₇₀₋₆₁₉ and EBNA₄₇₀₋₆₀₇, formed well-ordered crystals that diffracted beyond 2.5 Å resolution. In addition , this study also demonstrates the value of finding the most suitable piece of the protein for crystallization. This piece should fold into a compact domain for efficient packing into a crystal. Finding the optimal piece of the protein reduces the time spent searching for crystallization conditions. / Thesis / Master of Science (MS)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/23092 |
| Date | 04 1900 |
| Creators | Barwell, Jean |
| Contributors | Frappier, Lori, Biochemistry |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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