A targeting domain encoded by the yeast retrotransposon Ty5 that directs integration to silent chromatin

Thumbnail Image
Date
1999
Authors
Gai, Xiaowu
Major Professor
Advisor
Daniel F. Voytas
Committee Member
Journal Title
Journal ISSN
Volume Title
Publisher
Altmetrics
Authors
Research Projects
Organizational Units
Journal Issue
Is Version Of
Versions
Series
Department
Theses & dissertations (Interdisciplinary)
Abstract

The yeast Ty5 retrotransposon has a strong preference to integrate within silent chromatin at the telomeres and the silent mating-type loci. I developed an assay to monitor targeted integration of Ty5. Using this assay in mutant screens, I identified Ty5 targeting mutants with decreased target specificity to silent chromatin. The mutations clustered within a stretch of six amino acids, which define the Ty5 targeting domain. Site-directed mutagenesis experiments further confirmed the targeting domain's small size. When the targeting domain was tethered to a chromosome site, it nucleated the assembly of silent chromatin and resulted in transcriptional repression of adjacent marker genes. When overexpressed as part of a fusion protein, the targeting domain titrated away components of silent chromatin and resulted in the loss of transcriptional silencing at the telomeres. In separate experiments, I found that when Sir1p, a well-characterized recruiter of silent chromatin, was tethered to a plasmid, it created a Ty5 integration hot-spot. In summary, these experiments indicate that the Ty5 targeting domain interacts with components of silent chromatin. Tethering the Ty5 integration complex to silent chromatin by the targeting domain is likely the mechanism underlying target specificity. In addition, two Ty5 mutations that each cause an approximately six fold increase in transposition frequency were identified. Both mutations are in the gag gene and likely affect particle formation or RNA packaging.

Comments
Description
Keywords
Citation
Source
Subject Categories
Copyright
Fri Jan 01 00:00:00 UTC 1999