Endogenous Nuclear Retrotransposition Antagonists of Ty3, a Retroviruslike Element in Budding Yeast
Suzanne Sandmeyer, UC-Irvine
Biomedical and Clinical Sciences
Ty3 is a retroviruslike element in budding yeast which is studied as a model system for understanding the role of the host in supporting retroviruslike replication. A screen of 4500 yeast knockout mutants resulted in identification of 130 mutants affected (directly or indirectly) in Ty3 transposition. These were evenly divided between genes that positively and negatively affected transposition. Deletion of the checkpoint gene RAD24 negatively affected transposition, while deletion of a number of genes involved in DNA maintenance positively affected transposition. These include members of clamp loading complexes and proteins that interact with the replication clamp. Analysis of Ty3 protein and DNA intermediates in these mutants showed that in the case of a number of the DNA maintenance mutants the level of replicated Ty3 cDNA was increased. These data support a model where replicated cDNA may trigger a DNA damage response to Ty3 (as proposed for Ty1) which favors integration and perhaps repair of the integration site and that replication proteins may bind to and disrupt the Ty3 cDNA replication complex.
The laboratory is currently developing methods for ChIP analysis of Ty3 cDNA complexes. Using the yeast TAPtag collection in which individual epitope-tagged proteins are expressed from the native promoter, IgG beads will be used to precipitate various replication proteins and we will directly test for their associationwithTy3 cDNA by Southern blot analysis of precipitates. We are also developing methods using the nuclease benzonase to better understand the change in particle structure that accompanies cDNA synthesis and nuclear entry. This information will be used together with knowledge of bound replication proteins to develop a model for the basis of the antagonistic effect of replication proteins on Ty3 cDNA.
Because many yeast proteins have mammalian homologs, the results of this study will suggest host proteins that may also interact with retrovirus preintegration complexes and be natural restriction factors to retroviral replication.