The 2-micron circle of Saccharomyces cerevisiae is one of a small number of similar
DNA plasmids found only in budding yeast. To understand how this cryptic parasite persists,
despite conferring no advantage to the host, I investigated the plasmid-encoded Rep1 and
Rep2 proteins. Interaction of Rep1 and Rep2 with each other and with the plasmid STB locus
is required for equal partitioning of plasmid copies at mitosis. The Rep proteins also repress
expression of Flp, the recombinase that mediates plasmid copy-number amplification. In this
study, absence of Rep1 and Rep2, or over-expression of the plasmid-encoded Raf antirepressor,
increased expression of a longer, novel FLP transcript. Translation of this mRNA
may explain elevated Flp activity at low plasmid copy number. Raf competed for Rep2 selfassociation
and interaction with Rep1, suggesting the mechanism of Raf anti-repression.
Deletion analysis identified a target site for Rep protein repression of FLP that is also
repeated in the STB locus, suggesting this as the sequence required for Rep protein
association with both regions of the plasmid.
Distinct roles for Rep1 and Rep2 were identified; Rep1 was found to depend on Rep2
for post-translational stability, with Rep2 dependent on Rep1 for stable association with STB.
Lysine-to-arginine substitutions in Rep1 and Rep2 impaired their association with the host
covalent-modifier protein SUMO, suggesting these were sites of sumoylation. The
substitutions did not affect interaction of the Rep proteins with each other or their stability
but did perturb plasmid inheritance, suggesting that Rep protein sumoylation contributes to
their plasmid partitioning function. When Rep1 was mutant, both Rep proteins lost their
normal localization to the nuclear foci where 2-micron plasmids cluster, and were impaired
for association with STB, supporting this as the cause of defective plasmid inheritance. The
potential sumoylation-dependent association of the Rep proteins with the 2-micron plasmid
partitioning locus suggests the plasmid has acquired a strategy common to eukaryotic viral
and host genomes that depend on sumoylation of their segregation proteins for faithful
inheritance. Collectively, my results shed light on how the 2-micron plasmid maintains the
delicate balance of persisting without harming its host.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:NSHD.ca#10222/15863 |
| Date | 04 October 2011 |
| Creators | Pinder, Jordan Benjamin |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
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