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Download or read book Controlled Integration of the Ty5 Retrotransposon in Saccharomyces Ceverisiae [i.e. Cerevisiae] written by Junbiao Dai and published by . This book was released on 2006 with total page 218 pages. Available in PDF, EPUB and Kindle. Book excerpt: One essential step in the life cycle of retroelements is the stable integration of a copy of retroelement cDNA into the host genome. Random integration is potentially hazardous and could have deleterious genetic effects to the host. Therefore, elements and their hosts have coevolved mechanisms to regulate retroelement integration. In the budding yeast Saccharomyces cerevisiae, the Ty5 retrotransposon preferentially integrates into domains of heterochromatin. Targeting to these locations is determined by interactions between an amino acid sequence motif at the C-terminus of Ty5 integrase (the targeting domain) and the heterochromatin protein Sir4p. New Ty5 integration hotspots are created when Sir4p is tethered to ectopic DNA sites. Targeting to sites of tethered Sir4p is abrogated by single amino acid substitutions in either the targeting domain or Sir4p that prevent their interaction. Ty5 target specificity can be altered by replacing the integrase targeting domain with other peptide motifs that interact with known protein partners. Integration occurs at high efficiency and in close proximity to DNA sites where the protein partners were tethered. These findings indicate that Ty5 actively selects integration sites, and that targeting determinants are modular. These findings also suggest ways in which retroviral integration can be controlled, namely through addition of peptide motifs to viral integrases that direct integration complexes to specific chromosomal sites. The targeting domain of Ty5 integrase is post-translationally modified in vivo. Analysis by tandem mass spectrometry analysis revealed that the second serine within the targeting domain (S1095) is phosphorylated. Phosphorylation of the S1095 is required for targeted integration as measured by a plasmid-based targeting assay. Using surface plasmon resonance spectroscopy, S1095 phosphorylation was found to be required for productive interaction with Sir4C in vitro. This provides direct evidence for the requirement of post-translational modification in Ty5 targeting and reveals one mechanism that cells have adopted to overcome the deleterious effect of transposable element invasion; that is, they control integration specificity by modifying element-encoded proteins. Prompted by the discovery of targeting domain phosphorylation, a total of 109 kinase deletion strains were screened for kinases that affect Ty5 target specificity. Among eight candidates, the DNA damage checkpoint kinase Dun1p was found to be required, either directly or indirectly, for Ty5 transposition. The role of kinases in targeting was further explored by identifying which of the 109 non-essential protein kinases in S. cerevisiae affect the integrity of heterochromatin. Using transcriptional silencing as a measure of heterochromatin integrity, several kinases, including Dun1p, were identified that affect transcriptional silencing when mutated, principally at telomeres. Interestingly, most kinases are members of MAP pathways involved in DNA damage, osmolarity, cell wall integrity and pheromone responses. These results suggest that integrity of heterochromatin is tightly controlled by protein kinase cascades, which may indirectly contribute to the regulation of Ty5 integration specificity under different stresses. To determine which kinases directly phosphorylate Ty5 integrase, a portion of the integrase C-terminus (mINC) was purified and used as a substrate for in vitro kinase assays. All of the kinases encoded by the yeast genome (125 in total) were purified as GST-fusion proteins and tested their ability to phosphorylate mINC. Sixteen kinases were identified that modify mINC, and deletions in genes encoding these kinases affect Ty5 integration specificity. Interestingly, mutations in two kinases (ime2[delta] and rck2[delta] increased Ty5 integration specificity significantly. The remainder impaired integration specificity as measured by the plasmid-based targeting assay. Using several different mINC constructs with various serine/threonine mutations, the kinases were grouped based on their ability to phosphorylate a defined set of serine/threonine residues. Four kinases - Hrr25p, Rim11p, Rck2p and Yak1p - are the most likely candidates for phosphorylating S1095. Phosphorylation of mINC by multiple kinases, and importantly, the observation that mutations in these kinases affect Ty5 integration specificity, suggests that both transposition and integration of Ty5 are directly regulated in response to cellular processes or environmental stress.