Epstein-Barr virus nuclear antigen 1 (EBNA1) is the only viral product needed for replication of the latent Epstein-Barr virus genome. The latent origin of replication, oriP, consists of two cis-acting elements, the family of repeats (FR) containing twenty EBNA1 recognition sites, and the dyad symmetry element (DS) containing four recognition sites. Bidirectional DNA replication is known to occur within or near the DS. Previous studies have suggested that EBAN1 binds cooperatively to its recognition sites in the DS and have shown that EBNA1 binding induces DNA distortion within site 1 and site 4 of the DS. I have used EBNA1 mutants in electrophoretic mobility shift assays, methylation protection footprinting, and potassium permanganate reactivity analysis to examine EBAN1 assembly on the DS and the requirements for DNA distortion. I have found that: 1) EBNA1 has a 10-11 fold higher affinity for the outer two sites of the DS than the inner two sites due to DNA sequence variation, 2) the minimum region of EBNA1 necessary for site specific binding is contained within amino acids (a. A.) 470-607 but a.a. 459-470 greatly affect binding affinity, 3) EBNA1 dimers bind cooperatively on adjacent binding sites and the region responsible for this interaction is also contained between a.a. 470-607. I have also shown that EBNA1 binding to a single DS site 1 recognition site is sufficient to induce DNA distortion within that site and this distortion can be caused by a truncation mutant spanning a.a. 463-607 but not a.a. 468-607. Finally, although wild type spacing between recognition sites of the DS is critical for replication it is not crucial for EBNA1 binding or EBNA1 induced DNA distortion. / Thesis / Master of Science (MS)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/23175 |
| Date | 09 1900 |
| Creators | Summers, Heather |
| Contributors | Frappier, Lori, Biochemistry |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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