The yeast two-hybrid system was used to examine
interactions between the nine proteins involved in baculovirus DNA
replication. From the six proteins required for DNA replication,
four protein-protein interactions were identified, including an
interaction between LEF-1 and LEF-2, LEF-3 and itself, LEF-3 and
P143 (Helicase), and an interaction between IE-1 and itself.
The replication factors LEF-1 and LEF-2 interacted in both
yeast two-hybrid assays and glutathione S-transferase fusion
affinity assays. Using the yeast two-hybrid system, we mapped
the interaction domain of LEF-2 to amino acids between positions
20 and 60. Deletion analysis of LEF-1 failed to reveal an
interaction domain, suggesting that either multiple interaction
domains exists or the deletions disrupted secondary structures
required for the interaction. All of the deletions which were
unable to interact also failed to support significant levels of
transient DNA replication, suggesting that this interaction plays a
significant role in DNA replication.
The baculovirus single-stranded DNA binding protein, LEF-3, interacts with itself in yeast two-hybrid assays and glutathione S-transferase fusion affinity assays. Deletions of LEF-3, which were unable to interact with full length LEF-3, also failed to support transient DNA replication, suggesting that this interaction is required for the proper function of LEF-3. LEF-3 was purified to apparent homogeneity and analyzed by analytical ultracentrifugation, native PAGE and MALDI mass spectrometry, identifying the oligomeric structure of LEF-3 as a homotrimer.
In addition to interacting with itself, LEF-3 also interacts with P143 in yeast two-hybrid assays, immunoprecipitation experiments, and co-purification from a single-stranded DNA agarose column. The yeast two-hybrid system was used to map the LEF-3 interaction domain to the N-terminal 165 amino acids of LEF-3. Deletion analysis of P143 failed to reveal a delimited interaction domain. C-terminal deletions of LEF-3 containing amino acids 1 to 165 were unable to interact with full length LEF-3, indicating that the interaction of LEF-3 with itself (trimerization) is not required for the interaction between LEF-3 with P143. / Graduation date: 1998
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/33943 |
| Date | 17 February 1998 |
| Creators | Evans, Jay T. |
| Contributors | Rohrmann, George F. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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